Preparation of highly concentrated extracts from large volume of urine as the first step in detecting trace amounts of hypnotics in urine collected in drug-facilitated crime cases.

Purpose: Detecting hypnotics in victim urine samples collected several days after drug-facilitated crime (DFC) is challenging because most of the drugs have already been excreted. In this study, a sample preparation method was developed for extracting trace amounts of hypnotics using most of the urine excreted at one sampling time (100 mL), and large amounts of matrices were efficiently removed.

Methods: Etizolam, midazolam, ramelteon, and their metabolites were used as the target compounds. As the first step in decreasing the sample volume, solid-phase extraction using various sorbents was examined. The effects of additional clean-up columns (alumina, graphite, anion exchanger, etc.) on the removal of urine matrices were also examined. The pretreatment of 0.1-mL urine using a simple extraction column, specialized for small-scale urinalysis (Isolute Hydro DME +), was used as the reference method. The feasibility of drug detection in 100-mL urine was evaluated by comparison with a reference method.

Results: All analytes in 100-mL urine were most effectively adsorbed on a sorbent with octadecyl-bonded polymer and eluted with less than 2 mL of acetonitrile. A multilayer clean-up column consisting of alumina, octadecyl-bonded silica, and anion exchangers was effective in removing the matrices. α-Hydroxymidazolam was detected in 100 mL of urine that was collected 5 days after midazolam administration, but was undetected using the reference method.

Conclusions: This preparation method for 100-mL urine is useful as the first extraction step in detecting trace amounts of hypnotics in victim urine collected late after DFC.