Mitsuyoshi Kageura

New method of derivatization and headspace solid-phase microextraction for gas chromatographic–mass spectrometric analysis of amphetamines in hair

A simple method for hair analysis of methamphetamine (MAMP) and amphetamine (AMP) by gas chromatography-mass spectrometry (GC-MS) was developed using simultaneous headspace solid-phase microextraction (HS-SPME) with derivatization. After alkaline-digestion of hair, the analytes derivatized with heptafluoro-n-butyryl chloride were adsorbed on a polydimethylsiloxane-coated fiber by HS-SPME and analyzed by GC-MS. Their mass spectra were, respectively, observable at 1 ng per sample. The standard curves in the range of 0.1-100 ng were linear. The intra-day coefficients of variation at each 0.5 ng were 12.5% for AMP and 3.8% for MAMP. The applicability of this method was demonstrated in some case studies.

Highly sensitive and rapid determination of theophylline, theobromine and caffeine in human plasma and urine by gradient capillary high-performance liquid chromatography-frit-fast atom bombardment mass spectrometry

A sensitive and reliable analytical procedure has been established for the detection of theophylline (TH), theobromine (TB) and caffeine (CA) in human plasma and urine by gradient capillary high-performance liquid chromatography (HPLC)-frit-fast atom bombardment mass spectrometry (FAB-MS) (LC-frit-FAB-MS). Two capillary columns and a column-switching valve were used in this LC system to allow all of the sample injected to be introduced into the MS system. 7-Ethyltheophylline was used as the internal standard (I.S.). The xanthines in the specimen were extracted with an Extrelut column. The lowest detected amount was ca. 5 ng/ml using this method.

Headspace solid-phase microextraction and gas chromatographic-mass spectrometric screening for volatile hydrocarbons in blood.

Optimization for headspace solid-phase microextraction (SPME) was studied with a view to performing gas chromatographic-mass spectrometric (GC-MS) screening of volatile hydrocarbons (VHCs) in blood. Twenty hydrocarbons comprising aliphatic hydrocarbons ranging from n-hexane to n-tridecane, and aromatic hydrocarbons ranging from benzene to trimethylbenzenes were used in this study. This method can be used for examining a burned body to ascertain whether the victim had been alive or not when the burning incident took place. n-Hexane, n-heptane and benzene, the main indicators of gasoline components, were found as detectable peaks through the use of cryogenic oven trapping upon SPME injection into a GC-MS instrument. The optimal screening procedure was performed as follows. The analytes in the headspace of 0.2 g of blood mixed with 0.8 ml of water plus 0.2 microg of toluene-d8 at -5 degrees C were adsorbed to a 100-microm polydimethylsiloxane (PDMS) fiber for 30 min, and measured using the full-mass-scanning GC-MS method. The lower detection limits of all the compounds were 0.01 microg per 1 g of blood. Linearities (r2) within the range 0.01 to 4 microg per 1 g of blood were only obtained for the aromatic hydrocarbons at between 0.9638 (pseudocumene) and 0.9994 (toluene), but not for aliphatic hydrocarbons at between 0.9392 (n-tridecane) and 0.9935 (n-hexane). The coefficients of variation at 0.2 microg/g were less than 8.6% (n-undecane). In conclusion, this method is feasible for the screening of volatile hydrocarbons from blood in forensic medicine.

Identification of human hair stained with oxidation hair dyes by gas chromatographic-mass spectrometric analysis

This paper describes the gas chromatographic-mass spectrometric (GCMS) analysis of oxidation hair dyes from human hair. Diamines from the dyes were directly extracted from the hair in basic solution and aminophenols were extracted after neutralization. Both extracts were derivatised with trifluoroacetic anhydride and analysed by GCMS. Five components of oxidation hair dyes namely, p-phenylenediamine, toluene-2,5-diamine, o-aminophenol, m-aminophenol and p-aminophenol were clearly identified, whilst no other compounds originating from the hair dyes were detected. The presence and relative amounts of these dye components from hair extracts may assist in the discrimination of human hair especially in cases involving forensic science.

Demonstration of oxidation dyes on human hair

This paper describes a method of selected ion monitoring (SIM) analysis which can demonstrate the staining of human hair with oxidation hair dyes. Hair samples were decomposed with NaOH-Na2S2O4 solution by heating (100 degrees C, 30 min) in a stream of nitrogen. Basic and neutral ether extracts from the reaction mixture were trifluoroacetylated with trifluoroacetic anhydride in ethyl acetate and were then analyzed by SIM. The minimum lengths of a single hair for the detection of the 5 components of oxidation hair dyes acting as indicators were 1 mm for toluene-2,5-diamine, 2 mm for p-phenylenediamine, 20 mm for p-aminophenol, 50 mm for m-aminophenol and 100 mm for o-aminophenol. This method was applied to practical cases and the results were good.

High throughput chiral analysis of urinary amphetamines by GC-MS using a short narrow-bore capillary column

We report very rapid and simultaneous chiral analysis of urinary amphetamine-type stimulants (ATSs), including amphetamine, methamphetamine, 3,4-methylenedioxyamphetamine, 3,4-methylenedioxymethamphetamine, and 3,4-methylenedioxyethylamphetamine, using gas chromatography-mass spectrometry (GC-MS) with a simple procedure. A urine sample containing ATSs was subjected to extractive derivatization on a diatomaceous earth tube with trifluoroacetyl-l-prolyl chloride in a single step. The concentrated sample was analyzed by GC-MS, using a short narrow-bore capillary column (10 m × 0.1 mm i.d.) in split injection mode. All chiral isomers of the ATSs targeted in this study were chromatographically distinguishable within 5 min. By our method, ATSs in urine could be measured in the concentration range of 20–1000 ng/ml with coefficients of variation of less than 9%. Our method will be very useful for chiral analysis of ATSs in routine forensic toxicology investigations, because of its simplicity and rapidity.

Application of the drowning index to actual drowning cases

The drowning index (DI) was devised to diagnose drowning deaths, and is the weight ratio of the lungs and pleural effusion to the spleen. Among drowning (94 cases), mechanical asphyxia (47 cases), and acute cardiac (42 cases) deaths, within 2 weeks postmortem we compared six markers, the weight of each lung, pleural effusion weight, total weight of the lungs and pleural effusion, spleen weight, heart weight, and the DI. Statistical analysis revealed that the total weight was heavier, while spleen weight was lighter, and the DI was significantly larger in the drowning group (p<0.05). We examined the relation between the postmortem time and these markers. We divided 94 drowning cases into three groups according to the postmortem duration, group A (0-3 days; 43 cases), group B (3-7; 29 cases), and group C (7-14; 22 cases). The cut-off point of the DI was analyzed using the receiver operating characteristic (ROC) curve. As a result, the DI cut-off point was 14.1 in cases within two postmortem weeks. Drowning is still a difficult autopsy diagnosis, but in our experience, DI is a valuable indicator.

Practical GC/MS Analysis of Oxidation Dye Components in Hair Fiber as a Forensic Investigative Procedure

The purpose of this study was to improve the reliability of discrimination (or identification) of dyed hair by analyzing chemical substances present in the hair, as an addition to the conventional macroscopical and microscopical examinations and ABO blood group examination. Oxidation hair-dye components such as p-phenylenediamine (PPDA), toluylene-2,5-diamine (T-2,5-DA), o-aminophenol (OAP), m-aminophenol (MAP), p-aminophenol (PAP) and p-amino-o-cresol (PAOC) were selected as the object of study. After alkaline-digestion, hair samples were adjusted to a pH of 12.6 to 12.8, and applied onto an Extrelut column. After 15 min, the components were simultaneously extracted and derivatized with n-hexane including 1% heptafluoro-n-butyryl (HFB) chloride. Their HFB derivatives within a condensed sample were diluted in ethyl acetate, and analyzed by gas chromatography-mass spectrometry (GC-MS) with full mass scanning or selected ion monitoring. For estimating their levels, 2,4,6-trimethylaniline was used as the internal standard. Standard curves obtained by preparing a 5 cm segment of control hair spiked with authentic substances were linear, ranging from 0.1 to 4.0 micrograms for PPDA and T-2,5-DA, and from 0.01 to 0.4 microgram for OAP, MAP, PAP and PAOC. The coefficient of variation of inter-day precisions (each n = 4) was below 16% for PPDA, below 20% for OAP and PAP and below 24% for T-2,5-DA, MAP and PAOC. These components were detectable even at 6 ng for PPDA, T-2,5-DA, MAP and PAP, 8 ng for OAP, and 4 ng for PAOC. Recovery percents using this procedure ranged from 54 to 86%. By using actual dyed hair samples this method was shown to provide high sensitivity for accurate detection.

Two simple methods for enantiomeric analyses of urinary amphetamines by GC/MS using deuterium-labeled l-amphetamines as internal standards

Two simple methods for enantiomeric analyses of amphetamines in urine by gas chromatography-mass spectrometry (GC-MS) using l-amphetamine-d3 and l-methamphetamine-d6 as internal standards are presented. One method (method A) employs extractive derivatization on a diatomaceous column with (S)-(-)-N-(trifluoroacetyl)prolyl chloride (TPC) followed by separation with a conventional capillary column. The second method (method B) uses headspace solid-phase microextraction (HD-SPME) after derivatization with heptafluoro-n-butyryl chloride (HFB), followed by separation with an enantiomeric capillary GC column. By the two methods, all enantiomers were well separated in each chromatogram, and good linearity was obtained in practical concentration ranges (0.1–1.6μg/ml for method A and 0.05–1μg/ml for method B) for every compound by selected-ion monitoring. The precision studies indicated satisfactory coefficients of variation (<5%) for every enantiomer at 0.1μg/ml by both methods. Both methods were also evaluated by applying them to an actual poisoning case. Both methods are recommended for use in forensic analysis, because of their simplicity, high precision, and sufficient sensitivity.

Solid-phase microextraction for amphetamines in solid tissues: washing the homogenates with ethyl ether enables their measurements by GC-MS after heptafluorobutyryl derivatization

which the crude homogenate supernatant is washed with diethyl ether. D-Amphetamine (D-AMP), D-methamphetamine (DMAMP), L-AMP-d3 (IS-1), L-MAMP-d6 (IS-2), heptafl uorobutyryl chloride (HFB-Cl), and other reagents used in this study were the same as described in our previous reports [1,5]. Chicken liver was purchased from a local meat store for use in constructing a calibration curve. In an actual autopsy case, the liver, kidney, spleen, lung, brain, and blood were collected from a cadaver of a 29-year-old woman for analysis of amphetamines. The procedure established for analysis of amphetamines in solid tissue samples was as follows. Solid tissue (1.5 g) was incubated at 70°C for 2 h after the addition of 100 ng each of the two ISs and 5 ml of 0.1 M hydrochloric acid (this incubation under acid conditions softened the tissue and made it more suitable for homogenization), and then homogenized with a Polytron homogenizer. The crude supernatant fraction was obtained after centrifuging at 3000 rpm for 25 min. Two milliliters of the fraction was washed with 10 ml diethyl ether by shaking vigorously in a test tube or glassstoppered centrifuge tube. After standing for a few minutes, 1.0 ml of the aqueous phase was placed in a headspace vial, to which 0.5 g of sodium chloride, 0.2 ml of 1 M sodium hydroxide solution, and 0.01 ml of HFBCl were added. The septum-capped vial was shaken gently and set on a tray of an automated sampler for headspace-SPME (AOC-5000 Auto Injector, Shimadzu, Kyoto, Japan) followed by GC-MS analysis. The GC-MS analysis was performed on a QP-5000 instrument (Shimadzu) operated by GCMS-Solution (operation and data analysis system) using an Rtx-5 capillary column (10 m × 0.18 mm i.d., fi lm thickness 0.20 μm; Restek, Bellefonte, PA, USA). The GC oven Received: 1 October 2008 / Accepted: 17 October 2008 / Published online: 21 January 2009