A postmortem THC result that cannot be quantified because of matrix interference is not a negative test. Holland and colleagues encountered this problem despite using a validated two-dimensional GC–MS method. Their study shows why a report must distinguish an unusable measurement from a result below the quantification limit and from a valid numerical concentration.
The study selected cases by urine screening
The investigators studied 19 adult medical examiner cases, 16 male and three female, selected from deaths in Onondaga County, New York, during July–October 2009. Selection required an autopsy and a positive postmortem urine cannabinoid immunoassay. That screen selected cases; it did not quantify THC in blood or demonstrate impairment.
At autopsy, 10–15 mL cardiac blood and iliac vein blood were collected into labeled sodium fluoride tubes. Specimens were frozen at −60°C and shipped frozen. Analyses occurred two to ten months after collection. Twenty additional cannabinoid-screen-negative control cases supplied paired postmortem specimens for method evaluation. This was not a population survey of all deaths.
Validation supplied specific analytical limits
Following protein precipitation and solid-phase extraction, the method measured THC, 11-hydroxy-THC and THCCOOH using two-dimensional gas chromatography–mass spectrometry with cryofocusing. Detection limits were 0.25 ng/mL and quantification limits were 0.5 ng/mL for each analyte.
Calibration covered 0.5–50 ng/mL, with coefficients of determination of at least 0.990. Validation assessed quality controls at 0.7, 2, 20 and 40 ng/mL over four runs, totaling 16 replicates per level. Acceptance criteria included analytical recovery within 80–120% of target and imprecision no greater than 20% CV. These are method-performance criteria, not proof that every authentic decomposed specimen can be measured successfully.
Authentic specimens still caused failures
Putrefactive or in-vitro breakdown products markedly affected chromatography in some specimens. The investigators repeated analyses or reduced specimen volume to obtain acceptable results. In some specimens, poor internal-standard recovery persisted despite repeated attempts.
Table 1 reports two unquantifiable THC specimens in each matrix and one unquantifiable 11-hydroxy-THC specimen in each matrix. Table 2 identifies the corresponding paired cases. For THC, central blood had 11 quantifiable results and six below LOQ; peripheral blood had ten quantifiable results and seven below LOQ. Each matrix also had the two unavailable measurements. THCCOOH was quantified in all 19 cases at both sites.
The categories have different meanings. “Below LOQ” does not establish a concentration of zero, and an interference-related unavailable result does not establish either absence or a valid low concentration. A urine-positive case also need not have quantifiable parent THC in both blood specimens.
Dilution and repeat testing have defined limits
The researchers demonstrated dilution integrity up to 1:4 for THCCOOH in six specimens; the median difference from undiluted results was −3.0%, ranging from −13.2% to 7.3%. They could not perform equivalent dilution experiments for the other analytes because initial concentrations or specimen volumes were insufficient.
Successful THCCOOH dilution testing therefore does not automatically validate any dilution of THC or 11-hydroxy-THC. Repeat quantification supported reproducibility in the tested subsets, but did not rescue every failed specimen. Neither step establishes a concentration for a result that remained unavailable.
How to read the final laboratory report
Identify the analyte, matrix, reporting category and reason for nonreporting. Review internal-standard recovery, chromatograms, ion criteria, dilution records and the limits that apply to the actual method. Do not replace an unavailable measurement with zero when calculating a site ratio or summarizing detection rates.
The broader specimen problem is illustrated by vitreous screening compared with blood: a finding depends on both matrix and method. Even a valid quantified THC result is a chemical measurement, not an independent determination of impairment or cause of death.
Primary source
Holland MG, Schwope DM, Stoppacher R, Gillen SB, Huestis MA. Postmortem redistribution of Δ9-tetrahydrocannabinol (THC), 11-hydroxy-THC (11-OH-THC), and 11-nor-9-carboxy-THC (THCCOOH). Forensic Science International. 2011;212:247–251. doi:10.1016/j.forsciint.2011.06.028.
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