An assay that measures 82 drugs does not have identical quantitative performance for all 82, nor does validation in living-person whole blood automatically establish performance in every autopsy specimen. Steuer and colleagues’ multianalyte LC–MS/MS study reported six analyte exceptions and a specifically limited postmortem matrix assessment. Those details are more useful than the panel size alone when reviewing a result.
What the method was designed to do
The investigators developed a quantitative method using 200 µL of whole blood, protein precipitation, internal standards and an 18.5-minute LC–MS/MS run. Two multiple-reaction-monitoring transitions were acquired for each target. This was a defined targeted panel, rather than an unrestricted search for every possible toxicant.
Development and validation included blood from ten healthy volunteers and ten urine specimens. Ten previously drug-negative postmortem blood specimens were additionally examined for selectivity and matrix effects. Samples were stored at −20°C before their use in development or validation.
The paper did not provide one uniform postmortem blood collection site or death-to-collection interval. The autopsy specimens did not show severe putrefaction by the available case information or visual assessment. The experiment therefore should not be represented as comprehensive validation across heavily decomposed blood.
Which quantitative criteria had exceptions?
Using the selected internal standards and calibration models, 76 compounds met the specified bias and imprecision criteria: below 15%, or below 20% for the low quality-control level. Exceptions were buprenorphine, norclobazam, clonazepam, N-desalkylflurazepam, lormetazepam and triazolam.
The failures were level-specific. Buprenorphine failed the bias criterion in the above-calibration-range dilution experiment. The five benzodiazepines had bias outside the criterion at one of four quality-control levels. This is different from saying that every concentration for those compounds was unusable, or that the other 76 had failed.
Clonazepam illustrates why the table matters: at the low control concentration of 6 µg/L, bias was −25.7% and interday imprecision was 23.9%. A listed quantitation limit does not erase an observed failure at a tested control level. Review the relevant analyte and concentration, rather than relying only on the abstract’s general description.
What was tested in postmortem blood?
Postmortem selectivity and matrix-effect testing addressed interference and changes in instrument response. It did not repeat the entire living-blood quantitative validation at every concentration. The matrix-effect comparison used a medium quality-control level.
Across analytes, postmortem blood produced a mean additional suppression effect of 29% relative to living-person blood. This is an average instrument-response comparison, not evidence that 29% of a drug disappeared from a body or that every reported concentration should receive a 29% correction.
Internal-standard correction produced high reproducibility for all compounds in the tested postmortem specimens. That is useful evidence for this procedure and sample set. It does not establish equal absolute signal, successful performance in all decomposed specimens, or an unlimited ability to transfer the method to other matrices. A related benzodiazepine study using whole-blood calibration in autopsy tissues likewise shows why matrix-specific performance needs examination.
Detection limits and stability answer narrower questions
The method adopted analyte-specific lower quantitation limits from its calibration ranges. The authors did not systematically investigate limits of detection. A quantitation limit should therefore not be relabeled a separately established detection limit, and a negative panel result cannot exclude substances outside the panel.
Stability testing concerned processed extracts. Some absolute peak areas fell by more than 25% after 36 hours, while analyte-to-internal-standard ratios remained stable for 72 hours. This distinction concerns prepared material awaiting analysis. It does not demonstrate unchanged drug concentrations inside a body over a 72-hour postmortem interval.
How to assess an actual autopsy report
Identify the compound, quantitative range, control level, calibration model, dilution history and specimen condition. Check whether the reported number lies in a portion of the procedure supported by satisfactory performance. A mass-spectrometric identification and an accurately quantified concentration are related but separate claims.
Even a well-supported quantitative result does not alone establish dose, impairment, cause of death or the amount of postmortem redistribution. This study validates analytical operations within stated limits; it does not validate those broader biological inferences.
Primary source
Steuer AE, Keller M, Kraemer T, Poetzsch SN. Multianalyte Approach—Including Automated Preparation of Calibrators—for Validated Quantification of 82 Drugs in Whole Blood by Liquid Chromatography–Tandem Mass Spectrometry. Drug Testing and Analysis. 2025;17:954–988. doi:10.1002/dta.3794.
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