Simultaneous quantification of urea, uric acid, and creatinine in human urine by liquid chromatography/mass spectrometry.

In forensic analysis, the identification of urine or human urine among unknown liquids plays an important role. Urea, uric acid, and creatinine are major organic compounds found in human urine. Previous studies have reported that the concentration quotients of these three compounds can be used as an index for the identification of human urine. Here we describe a method for the simultaneous quantification of urea, uric acid, and creatinine in human urine by liquid chromatography/mass spectrometry (LC/MS), with the aim of forensic identification of human urine. Separation of the three analytes was achieved by hydrophilic interaction chromatography, using a TSK gel Amide-80 column with a mobile phase composed of acetonitrile and ammonium acetate aqueous solution, coupled with detection using a mass spectrometer. For quantification, melamine and violuric acid were used as internal standards. Human urine samples were pretreated for LC/MS analysis by dilution with LC mobile phase, followed by centrifugation and filtration. The analytes and internal standards were separated within 9 min. The linear ranges were 2.0-40.0, 0.10-1.60, and 0.13-2.00 mg/mL for urea, uric acid, and creatinine, respectively, with correlation coefficients > 0.99. The intra- and inter-day accuracies of the analytes were - 10.6% to 7.4%, and the precision was within 7.6%. For all analytes, no significant matrix effects were observed and recoveries ranged from 95.4% to 104.6%. Quantitative results of 3 analytes were obtained within their linear range from 10 human urine samples and the quotients, UA/UN × 20 and UA/Cre, were calculated based on previous reports.

Development of a dual test procedure for DNA typing and methamphetamine detection using a trace amount of stimulant-containing blood.

Investigation of drug-related crimes, such as violation of the Stimulant Drug Control Law, requires identifying the used drug (mainly stimulant drugs, methamphetamine hydrochloride) from a drug solution and the DNA type of the drug user from a trace of blood left in the syringe used to inject the drug. In current standard test procedures, DNA typing and methamphetamine detection are performed as independent tests that use two separate portions of a precious sample. The sample can be entirely used up by either analysis. Therefore, we developed a new procedure involving partial lysis of a stimulant-containing blood sample followed by separation of the lysate into a precipitate for DNA typing and a liquid-phase fraction for methamphetamine detection. The method enables these two tests to be run in parallel using a single portion of sample. Samples were prepared by adding methamphetamine hydrochloride water solution to blood. Samples were lysed with Proteinase K in PBS at 56°C for 20min, cooled at -20°C after adding methanol, and then centrifuged at 15,000rpm. Based on the biopolymer-precipitating ability of alcohol, the precipitate was used for DNA typing and the liquid-phase fraction for methamphetamine detection. For DNA typing, the precipitate was dissolved and DNA was extracted, quantified, and subjected to STR analysis using the AmpFℓSTR® Identifiler® Plus PCR Amplification Kit. For methamphetamine detection, the liquid-phase fraction was evaporated with N2 gas after adding 20µL acetic acid and passed through an extraction column; the substances captured in the column were eluted with a solvent, derivatized, and quantitatively detected using gas chromatograph/mass spectrometry. This method was simple and could be completed in approximately 2h. Both DNA typing and methamphetamine detection were possible, which suggests that this method may be valuable for use in criminal investigations.