Kunio Gonmori

Rapid nondestructive screening for melamine in dried milk by Raman spectroscopy

Melamine is a raw ingredient for the manufacture of plastics, but it is frequently misused by adding it to food to raise the nitrogen content, thereby giving the false impression of a high protein content. Varied amounts of melamine were added to samples of dried milk obtained from five manufacturers in Japan. The samples were illuminated by a small cross section of a laser beam and the scattered light was examined. The presence of melamine in milk could be immediately detected without any chemical or physical alteration of the milk by interpreting its Raman scattering spectra. Among the many Raman bands, an intense band at 676 cm−1 was the most useful for detecting melamine; the detection limit was about 1% (w/w). Because this method does not extract melamine from the dried milk sample, it reduces the risk of error that may occur during extraction or from interaction with chemical reagents. The method provides a very rapid screening test for melamineadulterated dried milk in food chemistry and forensic toxicology.

Simultaneous analysis of α-amanitin, β-amanitin, and phalloidin in toxic mushrooms by liquid chromatography coupled to time-of-flight mass spectrometry

An entire procedure for simultaneous analysis of α-amanitin, β-amanitin, and phalloidin in mushrooms by liquid chromatography (LC) electrospray ionization (ESI) time-of-flight (TOF) mass spectrometry (MS) has been optimized and established. We used a hydrophilic interaction column TSK-gel Amide-80 3 μm for LC separation, which enabled the simultaneous detection of the three toxins and internal standard microcystin RR. After homogenizing mushroom debris with methanol acidified with trifluoroacetic acid, the extract solution was subjected to solid-phase extraction with an Oasis HLB cartridge. The eluate was applied to the LC-ESI-TOF MS instrument. The calibration curves for the three toxins showed good linearity over the range of 100–1000 ng/g. The detection limits (signal-to-noise ratio = 3) for α-amanitin, β-amanitin, and phalloidin were about 30, 30, and 10 ng/g, respectively. The recovery rates of the three toxins at 100, 500, and 1000 ng/g were in the range of 53.1%–69.6%. The accuracy and precision (both intraday and interday) at 100, 500, and 1000 ng/g ranged from 87.9% to 117% and from 3.58% to 15.6%, respectively. Using the present method, the concentrations of the three toxins in the caps, stems, and roots of the toxic mushroom Amanita virosa were measured. The described method should be applicable to measurement of the Amanita toxins in human specimens, such as urine and blood of poisoned subjects.

Postmortem distribution of α-pyrrolidinobutiophenone in body fluids and solid tissues of a human cadaver

We experienced an autopsy case of a 21-year-old male Caucasian, in which the direct cause of his death was judged as subarachnoid hemorrhage. There was cerebral arteriovenous malformation, which seemed related to the subarachnoid hemorrhage. The postmortem interval was estimated to be about 2days. By our drug screening test using gas chromatography-mass spectrometry, we could identify α-pyrrolidinobutiophenone (α-PBP) in his urine specimen, which led us to investigate the postmortem distribution of α-PBP in this deceased. The specimens dealt with were right heart blood, left heart blood, femoral vein blood, cerebrospinal fluid, urine, stomach contents and five solid tissues. The extraction of α-PBP and α-pyrrolidinovalerophenone (α-PVP, internal standard) was performed by a modified QuEChERS (quick, easy, cheap, effective, rugged and safe) method, followed by the analysis by liquid chromatography-tandem mass spectrometry. Because this study included various kinds of human matrices, we used the standard addition method to overcome the matrix effects. The highest concentration was found in urine, followed by stomach contents, the kidney, lung, spleen, pancreas and liver. The blood concentrations were about halves of those of the solid tissues. The high concentrations of α-PBP in urine and the kidney suggest that the drug tends to be rapidly excreted into urine via the kidney after its absorption into the blood stream. The urine specimen is of the best choice for analysis. This is the first report describing the postmortem distribution of α-PBP in a human to our knowledge.

Mushroom toxins: a forensic toxicological review

Mushrooms are ubiquitous in the world. Amateur hunters harvest mushrooms growing in forests to enjoy eating them as seasonal delicacies, and occasionally they cause poisonings and even deaths. In this review, mushroom toxins are tabulated according to mushroom species, symptoms, toxicities and analytical methods on the basis of references. Second, because we constructed a method for analysis of amatoxins, the most virulent mushroom toxins, by liquid chromatography-time-of-flight-mass spectrometry, we introduce it for use in forensic toxicology. Third, an extensive poisoning incident after consumption of the usually edible mushroom Pleurocybella porrigens took place in nine prefectures in Japan from September to December 2004, resulting in 59 poisoned people including 19 deaths; this incident is briefly described and discussed in relation to its causative toxin(s). Finally, we present the chemical structures of new toxins purified from the highly toxic mushrooms Podostroma cornu-damae and Russula subnigricans; their structures were very unique, and the toxicities were comparable to those of amatoxins. From the forensic toxicological point of view, reports on sophisticated methodology for analyses of mushroom toxins seem to be too scant even for the well-known toxins. Hereafter, a number of toxic mushrooms and their new toxins are expected to be disclosed, especially because of environmental changes such as the global warming phenomenon.

Identification and quantification of metabolites of AB-CHMINACA in a urine specimen of an abuser

We experienced an autopsy case in which the cause of death was judged as poisoning by multiple new psychoactive substances, including AB-CHMINACA, 5-fluoro-AMB and diphenidine [Forensic Toxicol. 33 (2015): 45-53]. Although unchanged AB-CHMINACA could be detected from 8 solid tissues, it could neither be detected from blood nor urine specimens. In this article, we obtained eight kinds of reference standards of AB-CHMINACA metabolites from a commercial source. The AB-CHMINACA metabolites from the urine specimen of the abuser were extracted by a modified QuEChERS method and analyzed by liquid chromatography-tandem mass spectrometry before and after hydrolysis with β-glucuronidase. Among the eight AB-CHMINACA metabolites tested, only 2 metabolites could be identified in the urine specimen of the deceased. After hydrolysis with β-glucuronidase, the concentrations of the two metabolites were not increased, suggesting that the metabolites were not in the conjugated forms. The metabolites detected were 4-hydroxycyclohexylmethyl AB-CHMINACA (M1), followed by N-[[1-(cyclohexylmethyl)-1H-indazol-3-yl]carbonyl]-l-valine (M3). Their concentrations were 52.8 ± 3.44 and 41.3 ± 5.04 ng/ml (n=10) for M1 and M3, respectively. Although there is one preceding report showing the estimations of metabolism of AB-CHMINACA without reference standards, this is the first report dealing with exact identification using reference standards, and quantification of M1 and M3 in an authentic urine specimen.

Sensitive determination of arsenite and arsenate in plasma by electrospray ionization tandem mass spectrometry after chelate formation

Inorganic arsenite (As3+) and arsenate (As5+) are well-known poisons, and the toxicity of As3+ is about ten times that of As5+. In this study, a simple, rapid, and sensitive method was developed for As3+ in plasma using electrospray ionization (ESI) tandem mass spectrometry (MS-MS). After washing plasma with trichloroethylene (TCE), As3+ in the aqueous layer was reacted with pyrrolidinedithiocarbamate (PDC, C4H8NCSS-), and the produced As(PDC)3 was extracted with methyl isobutyl ketone (MIBK); a 1-µl aliquot of the MIBK layer containing As(PDC)3 was introduced into the MS-MS instrument in the direct-flow injection mode. Other arsenic compounds such as As5+, monomethyl arsonic acid, dimethyl arsinic acid, arsenobetaine, arsenocholine, and tetramethyl arsonium did not produce As(PDC)3. Therefore, without liquid chromatographic separation, As3+ alone could be detected after washing with TCE followed by solvent extraction of As(PDC)3 with MIBK. Thus, inorganic As5+ was reduced to As3+ with thiosulfate, and then the total inorganic As was quantifi ed as As3+; As5+could be calculated by subtracting As3+from the total inorganic As. The MS-MS quantification was performed by selected reaction monitoring using a peak at m/z 114 of a product ion (C4H8NCS)+ formed by collision-induced dissociation from the precursor ion As(PDC)2+ at m/z 367. The mass spectral identification on MS-MS spectrum was possible even at 1 ng As3+/ml plasma. The calibration curve for As3+ showed linearity from 0.5 to 100 ng/ml plasma. The limits of detection by selected reaction monitoring were 0.3 ng/ml in water and 0.2 ng/ml in plasma. The analysis could be completed in less than 15 min, because chromatographic separation was not necessary before the MS-MS detection.

Determination of Urine Luck in urine using electrospray ionization tandem mass spectrometry

A simple, rapid and sensitive method using tandem mass spectrometry (MS-MS) has been developed for the determination of chromate Cr6+ in urine. Cr6+ is a substantial component of Urine Luck, which is used to conceal the presence of drugs in urine. Cr6+ was complexed with diethyldithiocarbamate (DDC) and extracted with isoamyl alcohol in the presence of citric acid. Then a 1-μl aliquot of isoamyl alcohol containing Cr-DDC complex was directly injected into an MS-MS instrument without chromatographic separation. The quantification was performed using selected reaction monitoring at m/z 363.8 of product ion CrO(DDC)2+ obtained by collision-induced dissociation from the precursor ion, CrOH(DDC)3+ at m/z 513.1. This method was validated with the analysis urine samples obtained from volunteers. A linear calibration curve could be obtained in the range of 0.18–100 ng/ml. The limits of detection and quantification of Cr6+ were 0.05 and 0.18 ng/ml, respectively, using only 10 μl of urine. Results could be obtained in less than 10 min for a sample. After oxidation of Cr3+ to Cr6+, near 100% recovery was confirmed using standard reference materials such as SRM 2670a (low level and high level) and SRM 1643e. The most outstanding advantage of this ESI-MS-MS method is that it gives excellent product ion mass spectra for identification of Cr6+.

MALDI-TOF mass spectrometric determination of eight benzodiazepines with two of their metabolites in blood.

A rapid and sensitive method was developed for the determination of benzodiazepines and benzodiazepine-like substances (BZDs) by matrix-assisted laser desorption ionization (MALDI)-time-of-flight (TOF)-mass spectrometry (MS). In this method, α-cyano-4-hydroxy cinnamic acid was used as the matrix to assist the ionization of BZDs. Determination of 8 BZDs (with two of their metabolites) belonging to top 12 medical drugs detected in poisonous cases in Japan, was performed using diazepam-d5 as the internal standard. The limit of detection of zolpidem was 0.07ng/ml with its quantification range of 0.2-20ng/ml in blood, in the best case, and the limit of detection of flunitrazepam was 2ng/ml with its quantification range of 6-200ng/ml in blood, in the worst case. The spectra of zopiclone in MALDI-MS and MS/MS were different from those in electrospray ionization MS and MS/MS. Present method provides a simple and high throughput method for the screening of these BZDs using only 20μl of blood. The developed method was successfully used for the determination of BZDs in biological fluids obtained from two victims.

GC/MS with Post-Column Switching for Large Volume Injection of Headspace Samples: Sensitive Determination of Volatile Organic Compounds in Human Whole Blood and Urine

When volatile or semivolatile compounds are measured by headspace (HS) gas chromatography (GC)/mass spectrometry (MS), the maximum gas volume to be injected is usually 0.5-1.0 mL; over the volume, the MS detector automatically shuts down due to impairment of the vacuum rate of the MS ionization chamber. To overcome the problem, we modified the gas flow routes of a new type of GC/MS instrument to create a postcolumn switching system, which can eliminate the large volume of gas before introduction of target compounds into the MS ionization chamber. Our HS-GC/MS system enabled injection of as large as 5 mL of HS gas without any disturbance. As the first example analysis, we tried to establish the analysis of naphthalene and p-dichlorobenzene in human whole blood and urine by this method with large volume injection. The limits of detection for both compounds in whole blood and urine were as low as about 10 and 5 pg/mL, respectively. The validation data and actual measurements were also demonstrated. The new GC/MS system has great potential to analyze any type of volatile or semivolatile organic compounds in biological matrixes with very high sensitivity and full automation.

Platinum levels in various tissues of a patient who died 181 days after cisplatin overdosing determined by electrospray ionization mass spectrometry

Platinum (Pt) levels were determined in various tissues and body fluids obtained from a patient who died 181 days after cisplatin overdosing. The symptoms of cisplatin overdose, however, might have almost disappeared by day 40, and the patient’s death was ascribed to the recurrence of malignant lymphoma. Determination of Pt derived from cisplatin was performed by electrospray ionization mass spectrometry (ESI-MS) using silver (Ag) as internal standard. Pt and Ag complexed with diethyldithiocarbamate (DDC) in wetashed blood, and tissue solutions were extracted into isoamyl alcohol, and then acidified with oxalic acid. By injecting an aliquot of the isoamyl alcohol layer into a mass spectrometer in the direct flow injection mode, the quantitation was performed using the signals of Pt(DDC)3+ and Ag(DDC)2+ at m/z 639 and 403, respectively. The Pt levels ranged from 25ng/ml in blood to 2050ng/g wet weight in the liver of the patient, indicating that Pt remained at high levels in tissues, even after a period as long as 181 days after cisplatin overdosing.