Whole-blood calibrators can support quantitative work in alternative autopsy matrices when the complete procedure is evaluated, but an internal standard does not guarantee equal accuracy everywhere. A six-matrix benzodiazepine study reported generally acceptable performance alongside midazolam muscle bias near positive 30% and flunitrazepam negative bias up to 24%.
Why the investigators used one calibration matrix
Øiestad and colleagues analyzed peripheral blood, cardiac blood, pericardial fluid, psoas muscle, lateral vastus muscle and vitreous humor. Their selected case series contained 109 benzodiazepine- or z-hypnotic-positive cases from 183 project autopsies performed during June 2013–November 2016.
Calibrators and routine quality controls were prepared in whole blood obtained from a blood bank. The authors identified the use of whole-blood calibration for all six matrices as a limitation and said dedicated matrix calibrators would have been preferred. Restrictions on obtaining alternative matrices from routine cases constrained that approach.
They did not simply assume that blood calibration transferred correctly. Negative material from project cases was used to investigate matrix effects, extraction recovery, accuracy and precision in the alternative matrices.
Matrix effects and recovery were different tests
Matrix effects were evaluated at two concentration levels using specimens from five autopsy cases. Post-extraction-spiked samples were compared with neat standards; separately, samples spiked before extraction were compared with post-extraction-spiked samples to assess recovery.
Some ion suppression was observed, particularly in muscle. The authors reported that internal-standard correction addressed the observed suppression, with matrix-effect coefficients of variation below 15%. Extraction recovery was lower for several compounds in muscle. Recovery CVs were below 20%, except for diazepam in peripheral blood, where the reported CV was 26%.
Correcting ion suppression and obtaining consistent recovery are therefore not the same claim. Neither result by itself establishes that the complete concentration measurement has negligible bias.
The accuracy exceptions should remain visible
Most spiked-matrix QC results met the paper’s stated ±20% performance criterion. However, flunitrazepam showed systematic negative bias up to −24%, while midazolam muscle samples had positive bias close to 30%. A deviating low-level diazepam QC sample also affected precision and accuracy in peripheral blood, cardiac blood and pericardial fluid.
The main paper reported these exceptions alongside its overall favorable conclusion. Those analyte-specific findings limit how broadly the overall validation result can be applied: performance differed by compound and matrix.
The authors suggested that carbon-13-labeled standards might improve the affected measurements compared with deuterated standards. That was a proposed improvement, not a demonstrated correction tested in this case series.
Analytical performance did not validate a biological conversion
Specimens were collected at autopsy, generally within a week of death, then refrigerated at 4°C after laboratory arrival, typically for one to two weeks. Muscle was homogenized and dilution-corrected. Matrices from the same case were analyzed together as far as possible to reduce storage and instrument variation.
Even a successful calibration transfer would describe concentration in the collected specimen. It would not make muscle equivalent to femoral blood, reverse redistribution or establish a concentration during life. No antemortem comparison samples were available.
What a laboratory review should preserve
Identify the calibrator matrix, analyte-matched performance data, internal standard, recovery, matrix effects, bias and precision. Distinguish fortified QC experiments from authentic case measurements. Neither a well-fitted curve nor a positive drug result independently establishes impairment or cause of death.
For another calibration strategy and its limits, see standard addition in postmortem tissues.
Primary source
Øiestad EL, Øiestad ÅML, Middelkoop G, Brochmann GW, Thaulow CH, Vindenes V. Comparative Study of Postmortem Concentrations of Benzodiazepines and Z-Hypnotics in Several Different Matrices. Journal of Analytical Toxicology. 2023;47:287–298. doi:10.1093/jat/bkac106.
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