LC–MS Signal Suddenly Dropped or Disappeared: Root Causes, Diagnostics, and Fixes

LC–MS Signal Suddenly Dropped or Disappeared: Root Causes, Diagnostics, and Fixes
Comprehensive troubleshooting guide for ESI/APCI, Triple Quad, QTOF, and Orbitrap systems
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Understanding LC–MS Signal Loss
Keywords: LC–MS no signal, LC-MS signal loss, LC-MS sensitivity drop, ESI spray unstable, TIC flatline, MRM not detected, ion source contamination, divert valve to waste, ion suppression, vacuum fault, nitrogen gas issue
A sudden LC–MS signal drop (or complete disappearance) is almost always traceable to one of five areas: LC flow/plumbing, ion source/interface, MS tuning/acquisition, vacuum/detector hardware, or sample/matrix ion suppression. The fastest path to resolution is to isolate LC vs MS using a reference standard and a short, structured set of tests.
Scope and Symptom Definition
This troubleshooting guide addresses sudden loss of LC–MS signal, including:
No Peaks or Flat Baseline
No peaks, drastically reduced intensity, or a flat baseline (TIC or extracted channels)
Unstable Spray
Unstable spray, intermittent signal, or frequent source alarms
Instrument Coverage
Applicable to ESI and APCI sources across triple quadrupole, QTOF/Orbitrap, and single quadrupole instruments
Real Instrument Issues
Focused on real instrument/method causes (not data processing/integration artifacts)
Start Here
Rapid Triage to Locate the Failure
1) Determine Whether the Problem Is LC-Side or MS-Side
Perform a direct infusion of a known reference standard (tuning mix or a stable analyte standard):
Infusion normal → MS is fundamentally working; suspect LC flow, divert valve, plumbing, chromatography, or sample matrix
Infusion low/absent → suspect ion source, gas supply, vacuum, detector, interlocks, or wrong tune/method state
2) Check Instrument Status Messages and Interlocks Immediately
Review the instrument status panel/logs for:
HV disabled, source door open, missing cover, spray interlock
Vacuum faults, pump errors, or high background pressure
Gas supply low, nitrogen generator fault, dew point out of spec
These conditions can cause the instrument to auto-protect by disabling high voltage or limiting acquisition performance.
High-Probability Root Causes (and How to Prove Them)
1) Ion Source and Interface Failures (ESI/APCI)
Common Causes
Spray instability or loss (ESI): inadequate nebulizer/drying gas, incorrect temperatures, emitter/capillary blockage, contaminated cone/orifice, incorrect voltages
APCI sensitivity collapse: corona discharge instability, gas/temperature mismatch, contamination on the probe
Source HV off due to interlock or method state
Wrong polarity or wrong ionization mode (ESI vs APCI) in the method
Divert valve routing to waste during the analyte elution window
Diagnostics That Work Fast
Confirm source HV = ON and the correct polarity is selected
Verify nitrogen pressure, flow, and temperature setpoints
Inspect emitter tip / spray needle / transfer capillary for salt crust or particulates
Inspect cone/orifice for contamination films or deposits
Confirm divert valve timing: temporarily route the whole run to MS to test
Corrective Actions
01
Clean cone/orifice and relevant interface parts per vendor SOP
02
Replace/clean emitter and transfer capillary if restricted
03
Restore known-good source parameters (gas flows, temps, voltages)
04
Correct polarity/mode and verify the tune state used for acquisition
05
Fix divert valve program so the analyte window goes to MS, not waste
2) LC Flow, Degassing, and Plumbing Issues (Silent Signal Killers)
Common Causes
No flow or partial flow to the source (leak, empty bottle, pump cavitation)
Degasser failure introducing microbubbles → unstable spray and TIC spikes
Blocked frit/column/inline filter → unstable flow or altered split behavior
Post-column restriction or a change in tubing ID/length starving the MS
Unintentional split ratio change (or partial blockage in split capillary)
Diagnostics
Check pump pressure trace:
Sudden pressure drop
→ leak/no flow
Pressure oscillations/ripple
→ bubbles/cavitation/check valves
Sudden pressure increase
→ blockage/restriction
Confirm actual flow at the outlet (where feasible) and inspect for leaks at unions
Prime lines until flow is stable and bubble-free
Trace the entire path from column outlet → divert → source inlet (do not assume it matches yesterday)
Corrective Actions
Re-prime and purge solvent lines; confirm degasser status and vacuum behavior
Fix leaks; remake fittings; remove salt crystallization on connections
Replace inline filters/guard column; address blockages
Restore the correct post-column tubing and split configuration
If needed for stability, run briefly at lower flow and ramp up while watching pressure and TIC
3) Mobile Phase Composition and "Instant Ion Suppression"
A method can appear unchanged while the LC–MS response collapses due to a solvent or additive change.
Common Suppression Triggers
Non-volatile buffers (phosphate, sulfate) or high ionic strength
Detergents/surfactants (SDS, Tween)
High TFA (or sudden change in acid/base strength)
New solvent lot containing impurities or higher background contaminants
Diagnostics
Inject a clean standard prepared in a clean, volatile-compatible diluent
Compare response using a freshly prepared mobile phase with volatile additives
If a standard suddenly works after switching mobile phase lots, the root cause is likely composition/contamination, not the MS hardware
Corrective Actions
Replace non-volatile components with volatile buffers (ammonium formate/acetate)
Minimize or remove TFA when possible
Standardize solvent preparation, labels, and lot tracking
Flush the system to remove old additive films before resuming sequences
Key Insight: Ion suppression can mimic hardware failure. Always test with a clean standard in volatile mobile phase to differentiate matrix effects from true instrument problems.
4) Chromatography Changes That Hide the Peak (Even When the MS Works)
High-Frequency Failure Modes
Retention shifts move the analyte into a divert-to-waste window
Scheduled MRM windows are too tight after a column/temperature change
Column plugging or collapse causes broad, low peaks that look like "no signal"
Injection solvent mismatch causes breakthrough, splitting, or nonreproducible elution
Diagnostics
Temporarily send the entire run to MS (disable divert timing) and check for signal return
Widen scheduled retention windows (or temporarily disable scheduling)
Compare a standard in a clean matrix vs your sample
Check for sudden backpressure increase (blockage) or peak shape degradation
Corrective Actions
Update divert valve timing to match real elution
Widen scheduled windows and re-lock them after stabilization
Replace guard/column if pressure and peak shape indicate plugging
Match injection solvent strength to initial mobile phase; reduce injection volume if needed
5) Acquisition Method or Tune Configuration Errors
Common Causes
Wrong MRM transitions, wrong mass range, or disabled transitions
Incorrect collision energy or polarity mismatch
Too many MRMs → long cycle time → reduced dwell → apparent intensity collapse
Wrong tune file loaded (lens voltages or interface settings not appropriate)
Detector gain/EM settings limited or disabled
Diagnostics
Confirm precursor/product m/z, CE, polarity, and retention scheduling
Confirm scan range and resolution mode match your method intent
Load a known-good tune and compare critical voltages and lens settings
Check cycle time and dwell times against peak widths
Corrective Actions
Restore known-good method and tune
Reduce event count or improve scheduling to bring cycle time under control
Confirm detector settings are enabled and within expected ranges
6) Vacuum, Detector, and Hardware Faults
Common Causes
Vacuum instability after venting, pump faults, or leaks
Source door/cover interlocks disabling HV
Electron multiplier (EM) aging or auto-limiting behavior
Diagnostics
Check rough and high-vacuum readings and pump status
Confirm covers and interlocks are fully seated
Review detector gain/EM voltage and noise trends
Corrective Actions
If vacuum is out of spec or unstable, pause operation and follow your service escalation path
Clean source and interface, replace O-rings/gaskets where appropriate
Follow vendor qualification steps for EM performance and replacement when indicated
Critical Warning: Vacuum and detector issues require immediate attention. Do not attempt to continue operation if vacuum readings are abnormal or if detector performance is severely degraded.
Systematic Approach
Step-by-Step Diagnostic Workflow (Designed for Speed)
Step 1 — Confirm MS Health (Direct Infusion)
Infuse a known standard at a steady low flow.
Normal signal → proceed to LC-side checks
Weak/no signal → focus on source/gas/vacuum/tune/detector
Step 2 — Verify Source, Gas, and HV
Nitrogen supply pressure and dryness/purity are within spec
HV is on; correct polarity/mode selected
Clean/inspect cone/orifice and emitter/capillary
Step 3 — Confirm LC Flow and Degassing
Prime/purge until bubble-free
Inspect for leaks and restrictions
Review pressure trace for cavitation or blockages
Step 4 — Validate Divert Valve and Plumbing
Confirm analyte window goes to MS
Temporarily route everything to MS to rule out valve timing errors
Step 5 — Verify Acquisition Method and Tune
Confirm transitions, scan ranges, scheduling, cycle time
Reload known-good tune and compare settings
Step 6 — Evaluate Matrix Suppression vs True Instrument Loss
Inject a clean standard vs the real sample
If standards work but samples fail, suppression/cleanup is the main target
Step 7 — Maintenance/Service Escalation
Clean source/optics/ion optics per SOP
If vacuum/detector faults persist, escalate to service
Best Practices to Prevent Sudden LC–MS Signal Loss
Implementing these preventive measures will significantly reduce the frequency and severity of signal loss incidents:
Daily System Suitability
Run a daily system suitability check with a reference compound
Track Mobile Phase
Track mobile phase composition, pH, lots, and preparation notes
Use Volatile Buffers
Avoid non-volatile buffers; favor ammonium acetate/formate
Maintain MRM Windows
Keep scheduled MRM windows with safety margins and re-verify after any column/gradient change
Standardize Injection
Standardize injection solvent strength and volume
Regular Cleaning
Maintain a documented cleaning cadence for source and interface components
Key Takeaways
Summary
A sudden LC–MS signal drop is most commonly caused by ion source/interface contamination, gas/HV/interlock conditions, LC flow or degassing failures, divert valve misrouting, method/tune misconfiguration, matrix ion suppression, or vacuum/detector faults.
The fastest isolation strategy is a direct infusion check, followed by targeted verification of source conditions, LC flow stability, divert valve routing, and acquisition settings.