Masayuki Kashiwagi

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.

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.

Sudden unexpected death following stellate ganglion block

A 29-year-old woman, who had been suffering from left facial palsy, died 3.5 hours after undergoing stellate ganglion block (SGB). Autopsy revealed subcutaneous emphysema of the body, bilateral pneumothorax and a huge post-tracheal hematoma. The lower half of the trachea was markedly flattened by pressure from this hematoma. The cause of death was certified as airway obstruction due to a post-tracheal hematoma following SGB. The doctor cut both the anterior and the posterior wall of the trachea, because the trachea was flattened by the hematoma. He then intubated and sent air into the post-tracheal space. We consider that the patient could have recovered if she had received the correct tracheotomy. We therefore conclude that there was professional error on the part of the doctor in the form of medical malpractice.

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.

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.

Combination of a short middle-bore capillary column with a thicker stationary phase and a short narrow-bore separation column with a thinner stationary phase for the rapid screening of non-volatile drugs by gas chromatography–mass spectrometry

Dear Editor, An increase in requests for drug analysis has led to techniques that can analyze many samples in a short amount of time. For the purpose of enhancing the efficiency of analysis and increasing sample throughput, we looked at increasing the analysis speed of gas chromatography–mass spectroscopy (GC–MS), currently the most widely used technique for forensic drug analysis. The fast GC–MS techniques and the difficulty of their practical implementation have been reviewed in the literature [1]. Our group reported high-throughput chiral analysis of urinary amphetamines by fast GC–MS [2]. It seems very easy to establish a fast GC–MS method with a short narrow-bore capillary GC column and fast temperature programming, if using a modern GC–MS instrument that enables the rapid scanning of mass spectra and powerful pumping to maintain vacuum against the relatively large gas inflow into the ionization chamber. There are short narrow-bore capillary GC columns for fast GC–MS in the presence [3–5] and absence [2, 6, 7] of retention gaps, which are sometimes useful for obtaining narrow peak widths and symmetrical peak shapes. The retention gap is usually a short and deactivated capillary column without stationary phase. In this communication, we have tried to create a drug screening method by fast GC–MS using a short middle-bore capillary column with a thicker stationary phase in place of the inactive retention gap and a short narrow-bore capillary column. Our results showed that more than 20 drugs in whole blood could be screened within 4.1 min of GC–MS analysis. In this study, we mainly selected 30 psychotropic drugs [8, 9] as target compounds for analysis. Pure powder standards of fenfluramine-HCl, imipramine, diphenidolHCl, clomipramine-HCl, diazepam, chlorpromazine-HCl, nordiazepam, and trazodone were purchased from Sigma Aldrich (St. Louis, MO, USA); diphenhydramine-salicylate and nortriptyline-HBr from Wako (Osaka, Japan); and ethenzamide from Nacalai Tesque (Kyoto, Japan). Other drugs in the powder form were obtained from the corresponding pharmaceutical companies. Lidocaine-d10 was purchased from C/D/N Isotopes (Québec, Canada); diazepam-d5 and nordiazepam-d5 (both 1 mg/ml methanol solution) from Cerilliant (Round Rock, TX, USA). Caffeine-d3 (7-methyl-d3-theophylline) and 7-ethyltheophylline were synthesized in our laboratory. Other common reagents were of the highest purity commercially available. Blank whole blood was obtained from a healthy volunteer under permission; it was stored at -20 C until use. Methanolic stock solutions were made of the powder standards at a concentration of 1.0 mg/ml. A working stock solution of the 30 drug standards listed in Table 1 was made up in n-propyl acetate at a concentration of 20 lg/ml. Calibrators were prepared by adding enough of the standard working stock solution to 1 g of blank blood to yield the final concentrations of 0.10, 0.25, 0.50, 1.0, and 5.0 lg/ml in 16 9 125-mm screw cap tubes. A working internal standard (IS) solution containing caffeine-d3 (100 lg/ml), This article is for the special issue TIAFT2012 edited by Osamu Suzuki.

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

GC-PCI-MS/MS and LC-ESI-MS/MS databases for the detection of 104 psychotropic compounds (synthetic cannabinoids, synthetic cathinones, phenethylamine derivatives).

Designer psychotropic compounds continue to be a major problem in Japan and all around the world. Electron impact mass spectrometry (GC-EI-MS) and liquid chromatography with electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) data on these compounds have been widely reported. In this report, we present a detection method that has been rarely utilized to analyze these types of compounds, gas chromatography with positive chemical ionization and tandem mass spectrometry (GC-PCI-MS/MS). We report on the development of GC-PCI-MS/MS and LC-ESI-MS/MS databases of 104 psychotropic compounds, including 32 cannabinoid derivatives, 29 cathinone derivatives, 34 phenethylamine derivatives, and several other designer compounds. Using this database, we were able to detect 5 psychotropic compounds in an actual forensic autopsy case. If GC-PCI-MS/MS is used together with the more established methods of GC-EI-MS and LC-ESI-MS/MS, we believe the forensic toxicology community could be better prepared to deal with the challenges of these ever-changing compounds.

Development of a preparation method to produce a single sample that can be applied to both LC-MS/MS and GC-MS for the screening of postmortem specimens

Simple and efficient extraction methods have been developed for the screening of a wide array of drugs in postmortem autopsy specimens. Acidic and basic compounds were targeted with two extraction methods that can be applied to both GC-MS and LC-MS/MS instrumentation. The extractions were achieved by utilizing lipid-removal and solid-phase extraction cartridges while carefully monitoring the pH of the samples to ensure the adequate removal of interfering substances like lipids and amino acid derivatives. These methods were applied to actual autopsy cases, with 94 and 124 compounds detected by GC-MS and LC-MS/MS, respectively. The developed methods could easily be incorporated into a forensic laboratorys daily routine for screening many different compounds from postmortem samples.