Flunitrazepam and GHB are two substances commonly associated with DFC by the public, the media, and by healthcare professionals; however, flunitrazepam and its metabolite were detected in only one case of total submissions, calling into question its continued use as a DFC agent—especially since it has never been approved for medical use in the United States [8, 36]. Flunitrazepam is not only frequently discussed in media reports but also cited as one of the common substances of concern by the DEA as a DFC agent due to its short duration of action, short detection window, and impairing and amnestic effects [19]. Despite short detection windows (which may limit its detection due to a delay in reporting an incident), even ASB Standard 121 does not recommend including flunitrazepam in its general scope of analysis due to its low prevalence [9].

GHB is an endogenous substance commonly encountered in living individuals. Threshold guidelines set forth by ASB exist to attempt to separate endogenously produced GHB from exogenous administration. Various studies have suggested different thresholds for distinguishing between endogenous versus exogenous GHB, ranging from 5 to 20 mcg/mL [58, 59, 60, A. Eklund and M. Eriksson, Personal Communication, TIAFT, Helsinki (2000).]. Those studies also point out that analytical methods account for total GHB and GBL due to pH dependent equilibrium/interconversion [58]. The method used for the analyses reported does meet this requirement. Creatinine correction has been recommended in urine DFC cases to account for hydration or dehydration, which can be accounted for by normalizing the reported GHB value to creatinine [61]. Crookes et al. [60] recommend a GHB cutoff of 5 mcg/mL and a creatinine corrected GHB cutoff of 1000 mg/mmol (9.6 mg/g creatinine). While multiple threshold recommendations to distinguish between endogenous vs. exogenous GHB do exist, it appears that 10 mcg/mL is the established accepted threshold [10, 58, 59, 61]. There are additional factors that can cause increases in GHB levels that must be accounted for prior to interpreting the final GHB concentration. Warmer storage conditions (25°C) as well as freeze–thaw cycles, have caused increased GHB concentrations [9, 58, 59, 61]. Another situation that can cause increases in urinary GHB levels is GHB aciduria, which is an uncommon genetic disorder characterized by a succinic semialdehyde dehydrogenase deficiency that can increase endogenous urinary GHB concentrations to levels greater than 10 mcg/mL [58, 62].

Adopting a threshold recommendation of 10 mcg/mL in both blood and urine, <1% of GHB detections in either blood or urine in this study population suggest exogenous administration. Two cases in urine, after creatinine correction, changed interpretation for exogenous versus endogenous as the final reported result either increased above 10 mg/g creatinine (case 14) or decreased to exactly 10 mg/g creatinine (case 15) after correction, demonstrating the value of creatinine correction in urinary GHB analysis to account for urinary dilution due to dehydration or over hydration. Three cases contained between 10 and 25 mg/g creatinine, and two cases contained between 26 and 50 mg/g creatinine in urine. While these five cases met or exceeded the recommended GHB concentration threshold, no information was available regarding the disease states of the subject or the pH or storage conditions prior to reporting. In cases with GHB concentrations that make it challenging to differentiate between exogenous administration vs. endogenous levels, urine re‐collection at a later date has been suggested to establish an individual's baseline GHB concentration to provide additional context for interpretation [62]. If re‐collecting, additional results must be interpreted with caution as individuals experience baseline urinary GHB concentration fluctuations daily and at different time points [61]. However, if an individual's test results show a urinary GHB concentration between 10 and 25 mg/g creatinine, there may be some benefit in testing a later sample at the same collection time for additional context. Applying the recommended cutoff of 5 mcg/mL GHB and 9.6 mg/g creatinine proposed by Crooke et al. [60] results in the interpretation for all but two positive cases changing from exogenous to endogenous.

Two limitations to interpreting GHB concentrations in both matrices in this case series are that the collection interval and storage conditions prior to sample submission were not available; however, GHB was tested for in all cases. Urine samples were stored in frozen temperature conditions to help minimize preanalytical factors [59]. A nondetected GHB result should also be interpreted with caution, especially without understanding the full picture, as a negative result does not necessarily mean the substance was not present at the time of the incident, only that it was not detected when analyzed for.