LC–MS Signal Suddenly Dropped or Disappeared: Root Causes, Diagnostics, and Fixes (ESI/APCI, Triple Quad, QTOF, Orbitrap)

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, drastically reduced intensity, or a flat baseline (TIC or extracted channels)

• Unstable spray, intermittent signal, or frequent source alarms

• Applicable to ESI and APCI sources across triple quadrupole, QTOF/Orbitrap, and single quadrupole instruments

• 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

• Clean cone/orifice and relevant interface parts per vendor SOP

• Replace/clean emitter and transfer capillary if restricted

• Restore known-good source parameters (gas flows, temps, voltages)

• Correct polarity/mode and verify the tune state used for acquisition

• 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

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

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

• Run a daily system suitability check with a reference compound

• Track mobile phase composition, pH, lots, and preparation notes

• Avoid non-volatile buffers; favor ammonium acetate/formate

• Keep scheduled MRM windows with safety margins and re-verify after any column/gradient change

• Standardize injection solvent strength and volume

• Maintain a documented cleaning cadence for source and interface components

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.