In the analysis of tert-butyldimethylsilyl derivatives (IBDMS) of alkyl methylphosphonic acids (RMPA) and methylphosphonic acid (MPA), from soils by gas chromatography/mass spectrometry (GC/MS), the detection yields are generally low, due to the suppression of TBDMS derivatization by the soil matrix components and the adsorption of RMPA and MPA to the soils. An ion-exchange pretreatment of the aqueous soil extract can be used to overcome the former factor by removing interfering compounds. A pretreatment method is described for improving the detection yields due to the latter factor, using an alkaline extraction procedure. The recovery was estimated quantitatively using capillary electrophoresis. The soil samples tested included volcanogenous immature soils and showed a low aqueous extraction recovery and GC/MS detection yields. The inclusion of sodium hydroxide in the extraction solvent dramatically increased the recovery. Using a 0.1 M sodium hydroxide solution, the recovery was in excess of 68%. Interfering components were removed from the alkaline soil extract by solid-phase extraction of the acids on a silica-based strong anion exchanger. The alkaline soil extract was neutralized with hydrofluoric acid and applied to the cartridge in the fluoride form. After washing with water, MPA and RMPA could be eluted with methanolic ammonia nearly quantitatively. Using the established pretreatment method, MPA and RMPA were detected from all the soil samples in more than 67% yield.

The detectability and stability of lachrymators [2-chloroacetophenone (CN), o-chlorobenzylidene malononitrile (CS) and synthetic capsaicin (nonivamide)] were investigated using dichloromethane extraction followed by gas chromatography-mass spectrometry. Ultrasonication at 40°C slightly improved the extraction yields of nonivamide to almost quantitative yield, compared to low yields (30 to 40%) of CN and CS. In terms of the stability of low spiked concentrations (6 to 7 _g), from the surface of glass and stainless steel (60 to 100 cm square), CN rapidly disappeared, CS disappeared gradually, and nonivamide was almost quantitatively recovered. The three lachrymators in absorbent cotton (0.3 g) gradually disappeared. From water (20 mL), nonivamide gradually disappeared and was undetectable after eleven days. CN decreased gradually, and instead, acetophenone appeared. CS disappeared rapidly, and o-chlorobenzaldehyde, o-chlorobenzyl alcohol, and ochlorobenzoic acid were produced. The stability of high spiked concentrations of CN and CS in water (0.5 to 0.6 mg per 20 mL) was also investigated using liquid chromatography. CN was quantitatively recovered. The concentrations of CS decreased to about an8 % recovery level within 2 h. Concomitantly, o-chlorobenzaldehyde appeared at a concentration of about 80% recovery.

We established a method for determining capsaicin glucuronide in rat urine samples using liquid chromatography-mass spectrometry combined with enzymatic hydrolysis. Capsaicin was not detected in urine samples of rats administered capsaicin intraperitoneally (i.p., 2 and 4 mg/kg), but after hydrolysis with β-glucuronidase, extraction with methanol and solid-phase extraction, capsaicin was clearly detected by liquid chromatography-mass spectrometry with electrospray ionization. The limit of detection was obtained to be 0.1 ng/ml in urine sample matrix. The conditions for the enzymatic hydrolysis of the conjugate were optimized for β-glucuronidases from Ampullaria, Escherichia coli, bovine liver, Helix pomatia and Patella vulgata. The optimal conditions among those examined [pH (3.3-9.0), temperature (37-70°C) and enzyme amount (50-2500 U)] for 1 ml of the urine sample were as follows: β-glucuronidase from Ampullaria (pH 4.2, 45°C, 500 U), from Escherichia coli (pH 6.0-7.2, 37°C, 500 U), from bovine liver (pH 5.0, 45°C, 500 U), from Helix pomatia (pH 5.0, 60°C, 1250 U) and from Patella vulgata (pH 3.8, 45-60°C, 2500 U). Among the enzymes examined, β-glucuronidase from Ampullaria was found to be suitable for hydrolysis of the conjugate. Under the optimal conditions of Ampullaria β-glucuronidase, the incubation of 1 ml of urine sample for 90 min was sufficient for hydrolysis of capsaicin glucuronide in the urine sample. The urinary recovery values of capsaicin collected for 0-48 hr after administration of 2 and 4 mg/kg capsaicin to rats were 1.4 and 1.1%, respectively.Key words ---capsaicin, glucuronide conjugate, enzymatic hydrolysis, β-glucuronidase, liquid chromatographymass spectrometry, urine mainly metabolized into compounds and their glucuronides (Fig. 1) 7,8) and that cytochrome P-450 converted capsaicin to various oxidized compounds, 9) the details of the in vivo metabolism of capsaicin and related compounds has not yet been fully elucidated. To prove exposure to pepper spray in forensic analysis, it is required to detect capsaicin and related compounds from evidence samples, especially from biological samples of casualty cases.We previously reported on a quantification method for urinary capsaicin and its hydrolyzed compound, vanillylamine.10) The analysis of their glucuronides in urine samples is also important for forensic analysis, as they are major metabolites in urine. 7,11)