Carryover in LC-MS/MS Forensic Toxicology: Detection, Prevention, and Case Review

Carryover in forensic toxicology is the unintended appearance of analyte signal in a later injection because material from an earlier sample remains somewhere in the analytical system. In LC-MS/MS and related mass-spectrometric workflows, a very high-concentration specimen can leave residual analyte in the autosampler needle, injection path, valve, tubing, column, source, or another wetted surface. If that residual material is released into the next injection, the resulting signal can mimic or inflate a true low-level finding.

Carryover is therefore not merely an instrument-maintenance issue. It is a method-validation, batch-review, sequence-design, and case-interpretation issue. A scientifically defensible review asks not only whether a laboratory validated carryover, but also whether the concentration range used in validation was appropriate for the specimen immediately preceding the questioned result and whether the laboratory’s batch records show that its carryover controls were actually satisfied.

Why carryover matters in forensic casework

The practical importance of carryover is greatest when a low or borderline result follows a specimen containing a much larger amount of the same analyte. The concern is not that every low result is carryover; it is that the sequence context becomes part of the analytical evidence.

• A high-concentration positive may contaminate the injection pathway.
• The following blank or case specimen may show residual signal.
• The magnitude of that signal may be analyte-specific and instrument-specific.
• Carryover may matter more near a reporting limit, cutoff, or other consequential decision point.
• Reinjection alone may not distinguish a true specimen finding from contamination introduced during the original sequence.

This is why sequence position, blanks, wash steps, reinjection history, re-extraction, and the concentration of the preceding specimen can be material to technical review. For a focused case-review workflow, see Injection Sequence Review in Forensic Toxicology.

Carryover is a required validation consideration

ANSI/ASB Standard 036, Standard Practices for Method Validation in Forensic Toxicology, establishes minimum practices for validating forensic toxicology methods and is intended to demonstrate that a method is fit for its intended use. Carryover is one of the performance characteristics routinely evaluated in modern toxicology validation studies.

A useful carryover study challenges the system with a concentration high enough to reflect the laboratory’s realistic upper casework range and then examines a blank or negative specimen immediately afterward. The laboratory should define what level of residual response is acceptable and what action is required when the criterion is exceeded.

The key question is not simply, “Was carryover tested?” It is: Was it tested under conditions that meaningfully bracket the case being reviewed?

How laboratories evaluate carryover

A typical validation design places a negative specimen after a high-concentration positive challenge. The blank is then examined for analyte response. Published forensic methods illustrate several legitimate ways laboratories implement this concept, but the exact acceptance criterion is method-specific.

For example, a 2025 Journal of Analytical Toxicology LC-QTOF-MS validation for 946 drugs and metabolites evaluated blood and urine blanks after high-concentration quality controls and after samples at five times the high-control concentration. The blank injections were assessed for responses above the method’s limit of sensitivity. Sarkisian and Rodda, 2025.

Another forensic LC-MS/MS validation for Δ8- and Δ9-THC compounds challenged the system with standards at concentrations of at least 1000 ng/mL and required the following extracted negative samples to remain below a defined fraction of the lowest calibrator signal. See the published cannabis-isomer validation.

These examples should not be converted into a universal numerical rule. They instead demonstrate the proper analytical principle: carryover must be evaluated using a predefined, validated decision criterion appropriate to the particular method.

Why the preceding specimen concentration matters

Suppose a method showed acceptable carryover after a validation challenge at 100 ng/mL, but a questioned case specimen followed a sample containing 5,000 ng/mL of the same analyte. The original validation may still provide useful information, but it does not automatically establish how the system performs after a challenge fifty times larger.

This becomes especially important for compounds that adsorb to surfaces, have poor washout characteristics, produce very large detector responses, or are encountered in casework over an unusually wide concentration range. A defensible review therefore compares the concentration of the preceding positive with the concentrations actually used during validation and routine carryover checks.

Carryover can be analyte-specific

Not all analytes behave the same way. A 2024 Journal of Analytical Toxicology method covering more than 250 novel synthetic opioids and xylazine reported measurable carryover for several analytes after high-concentration specimens. The laboratory used a strong needle-rinse mixture and required re-extraction of samples following sufficiently high positives for the affected compounds. See the published method.

This illustrates an important forensic principle: a method can be fit for purpose even when certain analytes require additional precautions. What matters is whether the laboratory characterized the limitation, adopted an appropriate control strategy, and followed that strategy in the case under review.

Where carryover can originate

• Autosampler needle or needle seat: residual sample may remain after injection.
• Injection loop and valve: analyte can persist in internal wetted surfaces.
• Tubing and fittings: adsorption and delayed release can produce memory effects.
• Chromatographic column: strongly retained material can elute in later runs.
• Ion source: contamination can contribute to persistent background or delayed analyte release.
• Sample-preparation workflow: contamination can also arise before injection through pipettes, wells, extraction hardware, or preparation sequence.

Instrument carryover and pre-analytical cross-contamination are related but not identical problems. A case review should not assume that a clean instrument blank excludes every possible contamination pathway.

Carryover versus matrix effects and interference

Carryover should also be distinguished from matrix effects and analytical interference. Matrix effects alter ionization efficiency; interference can create or distort a signal because another substance behaves similarly in the analytical method. Carryover, by contrast, involves residual material from an earlier analytical event.

The distinction matters because the corrective investigation is different. A laboratory troubleshooting carryover may inspect sequence order, wash solutions, needle-rinse settings, blanks, reinjections, and maintenance. A laboratory investigating matrix effects may instead evaluate suppression/enhancement, internal-standard behavior, extraction recovery, and matrix matching.

What should happen when carryover is suspected?

A reasonable investigation may include one or more of the following, depending on the validated laboratory procedure:

1. Review the injection sequence and identify the immediately preceding samples.
2. Compare the preceding concentration with the method’s validated carryover challenge.
3. Examine intervening blanks, negative controls, and wash injections.
4. Review chromatograms, transition ratios, retention time, and internal-standard behavior.
5. Reinject an existing extract if that addresses an instrument-sequence question.
6. Perform an independent re-extraction when necessary to distinguish an extract/injection issue from a specimen-level finding.
7. Document maintenance or wash changes if the instrument required corrective action.
8. Apply the laboratory’s validated decision rule before reporting the result.

Reinjection and re-extraction answer different questions. A reinjection tests the already-prepared extract again; a re-extraction repeats more of the analytical process from the original specimen. For additional context, see Measurement Uncertainty Near Forensic Toxicology Cutoffs and Technical and Administrative Review in Forensic Toxicology.

Case-review questions for a low-level result

• What specimen was injected immediately before the questioned sample?
• Did that specimen contain the same analyte?
• At what concentration?
• What concentration was used to challenge carryover during validation?
• Was a blank injected between the high positive and the questioned sample?
• What did that blank show?
• What carryover acceptance criterion was in force on the date of analysis?
• Did the laboratory have analyte-specific rules for extreme positives?
• Were additional washes, reinjections, or re-extractions required?
• Were those steps documented in the batch record?
• Was the questioned result near the LOD, LOQ, cutoff, or reporting threshold?
• Did technical review specifically evaluate the sequence context?

What carryover does not prove

The mere theoretical possibility of carryover does not prove that a reported result is false. Conversely, a laboratory’s statement that its method was validated does not establish that carryover is impossible in every future sequence. The scientifically appropriate approach is case-specific: compare the actual sequence, concentrations, blanks, controls, method validation, corrective-action rules, and raw analytical data.

Frequently asked questions

Can LC-MS/MS carryover cause a false positive?

It can create residual analyte signal in a later injection if the method’s washout and sequence controls are inadequate for the preceding concentration. Whether that residual signal would meet the laboratory’s identification and reporting criteria is method- and case-specific.

Does a blank after a high positive solve the problem?

A properly placed blank is important evidence because it directly challenges residual signal after the preceding injection. But the interpretation still depends on the analyte, the concentration challenge, the blank acceptance criterion, the method’s sensitivity, and whether contamination could have arisen earlier in sample preparation.

Is carryover more important for low results?

Usually. A small residual response is more likely to be consequential when the questioned result is itself small, especially near a detection, quantitation, reporting, or legal decision threshold.

Can a re-extraction help evaluate suspected carryover?

Yes. An independent re-extraction can provide information that a simple reinjection cannot because it repeats specimen preparation and creates a new extract. The appropriate response should follow the laboratory’s validated procedure and the specific reason carryover is suspected.

Technical takeaway

Carryover is best evaluated as a sequence-specific analytical question, not as an abstract possibility. The strongest review compares the actual preceding specimen concentration with validated carryover challenges, examines blanks and controls, checks analyte-specific corrective rules, and determines whether the laboratory followed those rules. That approach separates a scientifically supported carryover concern from speculation while protecting against overconfidence in a low-level result simply because the instrument produced a number.