Can a Validated Blood EtG/EtS Assay Prove Drinking Occurred Before a Crash?

No. Validating a blood EtG/EtS assay establishes how well a laboratory method measures alcohol metabolites; it does not by itself establish whether drinking occurred before or after a crash. A 2021 study developed a rapid whole-blood method with good analytical performance. Its findings support careful metabolite measurement, while leaving the timing and impairment questions dependent on additional evidence.

Ethyl glucuronide (EtG) and ethyl sulfate (EtS) are ethanol metabolites, not ethanol itself. Their detection can add information to an alcohol investigation, but a positive metabolite result is not a measured BAC or a direct measure of driving ability.

What the primary study validated

Sidqey and colleagues developed an ultra-high-performance liquid chromatography–tandem mass spectrometry method using 100 microlitres of antemortem or postmortem whole blood. Protein precipitation was followed by phospholipid removal, with a 4.5-minute analytical cycle.

The method used deuterated internal standards and separate mass-spectrometric transitions for identification and measurement. The reported calibration ranges were 0.089–22 mg/L for EtG and 0.025–6.3 mg/L for EtS. These concentrations describe metabolites in whole blood, not urine cutoffs, serum ethanol or breath alcohol.

The study primarily assessed analytical performance and routine sample results. It did not administer a controlled before-versus-after-crash drinking protocol or validate a classifier resolving a particular post-incident drinking claim.

Detection, quantification and reporting are different thresholds

The reported limits of quantification were 0.067 mg/L for EtG and 0.019 mg/L for EtS. The laboratory’s positive reporting cutoffs corresponded to the lowest routine calibrators, which were higher. A method can therefore detect or quantify a small amount without reporting it as positive under the laboratory’s chosen decision rule.

A negative report must be interpreted against the actual cutoff and procedure. It does not necessarily mean the specimen contained no molecules of the metabolite. Likewise, the validation’s analytical sensitivity cannot be treated as the sensitivity of a test for impairment or pre-crash drinking.

Why cleanup and internal standards mattered

Whole blood contains substances that can affect ionization and chromatography. The investigators compared cleanup approaches because protein precipitation alone could leave problematic material, particularly in decomposed specimens.

Without internal-standard correction, the measured matrix responses were suppressed. After correction, reported matrix effects were approximately 99–103%. Extraction recovery was at least 61% for EtG and 77% for EtS. Recovery below 100% does not mean the final reported concentration must be multiplied by a simple loss factor: calibration and internal-standard procedures are part of quantification.

Between-assay precision was no worse than 4.5% in the reported validation measurements, with bias ranging from −15% to +8%. These figures describe performance under specified conditions, not guaranteed error bounds for every future specimen.

Authentic-sample comparisons revealed limits as well as strengths

Comparison with the former method used 120 forensic autopsy samples. General agreement was good, but a significant negative bias appeared, especially toward the higher measurement range. The authors discuss improved chromatography and calibration-range issues; it would be inaccurate to summarize the methods as numerically identical.

Longer-term routine comparisons estimated precision of no worse than 5.1% in antemortem samples and 6.7% in postmortem samples. The approximately 6,000 analyses described in the report included replicate analyses and corresponded to about 3,000 individual samples. They were not 6,000 independent drinking experiments.

What the postmortem observations can establish

Among 747 ethanol-positive postmortem cases examined over 19 months, EtG was detected in 571 and EtS in 589. Approximately 17% lacked EtG and/or EtS above the study’s stated half-cutoff criterion. The authors considered this potentially indicative of postmortem ethanol formation.

“Potentially indicative” is not proof that every such ethanol result arose from fermentation. Interpretation still requires circumstances, specimen condition, storage, timing and other analytical evidence. Those postmortem proportions also cannot be applied directly to living drivers.

Applying the method evidence to DUI claims

A reliable metabolite measurement is a necessary foundation for interpretation, not the entire interpretation. Establish the whole-blood specimen, collection time, cutoff, calibration range, quality controls and uncertainty before considering the drinking timeline.

The separate EtG/EtS timing-ratio study concerns biological timing models. This validation paper concerns measurement capability. Neither a positive metabolite test nor the fact that an assay is validated automatically establishes incident-time BAC, impairment, crash causation or that a post-crash drinking account is false.

Primary source

Sidqey D, Liane VH, Kristoffersen L. Quantitative Determination of Ethyl Glucuronide and Ethyl Sulfate in Postmortem and Antemortem Whole Blood Using Phospholipid Removal 96-Well Plate and UHPLC–MS-MS. Journal of Analytical Toxicology. 2021;45:378–388. https://doi.org/10.1093/jat/bkaa108.