Blank specimens and fortified controls can miss an interference present only in authentic samples. Toennes and colleagues discovered such a problem while developing a serum LC–MS/MS cannabinoid method. Their study shows why selectivity must be demonstrated in real specimens, while providing no basis to assume that every postmortem cannabinoid result is wrong.
This was serum-method research, not an autopsy cohort
The investigators studied THC, 11-hydroxy-THC and THCCOOH in human serum. During method development, authentic cannabis-user specimens showed a shoulder on the THCCOOH chromatographic peak that was absent from blank and fortified specimens.
The extraction used 0.2 mL serum, internal standards and solid-phase extraction before LC–MS/MS. The paper did not examine decomposed autopsy whole blood or define anatomical collection sites and postmortem intervals. Its relevance to postmortem work is the analytical question of selectivity; it does not supply direct estimates of interference frequency in autopsy specimens.
Two monitored transitions did not eliminate the interference
The unknown compound shared the THCCOOH precursor mass and both monitored fragment transitions, but had different relative fragment intensities. The investigators could not establish selective transitions that distinguished the two compounds on their own.
With an extended chromatographic program, THCCOOH eluted at 5.11 minutes and the interfering compound at 5.63 minutes. Separation allowed examination of each peak. The authors proposed structural similarity to THCCOOH, possibly an isomer, but had not established the compound’s exact structure or origin. It should not be retrospectively given a definitive chemical identity on this paper alone.
The reported percentages were estimates with a specific denominator
Of 98 forensic samples analyzed, 93 were evaluated after excluding four with no detected cannabinoids or interference and one with THCCOOH below quantification. Twenty more were excluded from the relative-concentration evaluation because the low interfering signal could not be integrated adequately.
In the remaining 73, estimated interference-to-THCCOOH concentration ratios ranged from 4.5% to 51.0%, with a median of 14.6%. The estimation used THCCOOH calibration and summed fragment responses, not a pure reference standard for the unknown compound. These percentages therefore are not demonstrated correction factors for unrelated laboratory results.
The estimated concentrations were strongly associated with THCCOOH (Spearman coefficient 0.883, P < 0.001). Association with cannabis-positive specimens did not resolve whether the compound was a metabolite or another related substance.
Method optimization addressed the observed problem
The final optimized method separated the interference while retaining a short analytical cycle. Reported quantification limits were 0.3 μg/L for THC, 0.2 μg/L for 11-hydroxy-THC and 0.6 μg/L for THCCOOH. Calibration extended to 50 μg/L for the first two and 150 μg/L for THCCOOH.
Reported recovery exceeded 95%; within-day and between-day precision was below 11%, and bias below 5%. Comparison of THCCOOH measurements using the optimized and extended methods in 93 samples showed mean deviation of 0.01% and relative standard deviation of 2.6%. Thus the study documented an identified problem and an effective response, rather than failure of LC–MS/MS as a technology.
What to examine in a postmortem laboratory record
Ask whether the actual method separates relevant interferents, how retention times and ion ratios were evaluated, and whether authentic specimens of the relevant matrix were included in validation. A clean fortified control cannot by itself establish selectivity in every real specimen.
An interference can affect identification or quantitation without being postmortem redistribution. Conversely, valid chemical measurement does not establish impairment, time of use or cause of death. See unquantifiable postmortem THC results for a different study in which authentic autopsy matrices directly limited reporting.
Primary source
Toennes SW, Hanisch S, Pogoda W, Wunder C, Paulke A. Pitfall in cannabinoid analysis—detection of a previously unrecognized interfering compound in human serum. Analytical and Bioanalytical Chemistry. 2015;407:463–470. doi:10.1007/s00216-014-8301-y.
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