Kayoko Minakata

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.

Determination of iodide in urine using electrospray ionization tandem mass spectrometry

A rapid and sensitive electrospray ionization (ESI) tandem mass spectrometry (MS-MS) procedure was developed for the determination of iodide (I(-)). A gold (Au) and I(-) complex was formed immediately after the addition of the chelating agent NaAuCl(4) to I(-) solution, and was extracted with methyl isobutyl ketone. One to five microliters of the extract were injected directly into an ESI-MS-MS instrument. I(-) quantification was performed by selecting reaction monitoring of the product ion I(-) at m/z 127 derived from the precursor ion (197)AuI(2)(-) at m/z 451. I(-) concentration was measured in the quantification range from 10(-7) to 10(-5) M using 50 microL of solution within 10 min. Iodate was reduced to I(-) with ascorbic acid and determined. I(-) concentration in reference urine 2670a was measured after treatments.

A NEW COLORIMETRIC DETERMINATION OF DIQUAT PRODUCED WITH SEVERAL MODERATE REDUCTANTS

A new colorimetric method is described for the quantification of diquat. A red colored compound is produced on the reduction of diquat with either 2-mercaptoethanol, dl-dithiothreitol or l-cysteine in 0.2 N NaOH solution. The absorption maximum of the red colored compound is 495 nm and the molar absorption coefficient is 2.072 × 104 (0.1125 in 1 μg diquat/ml with 1 cm light path). The red color is clearly visible in 5.43 × 10−6 M solution (= 1 μg/ml) suggesting that this reaction is applicable as a color test of diquat. The absorption at 495 nm shows a linear concentration dependence for the range 0.1 – 10 μg diquat/ml. The fading of the color is less than 5% after 1 h. Under the same condition, paraquat does not produce any colored products. Therefore, in a mixed solution containing both diquat and paraquat, the concentration of diquat can be determined at first from the absorption at 495 nm after reduction with 2-mercaptoethanol. Then by adding sodium dithionite to the solution, the concentration of paraquat is calculated by subtracting the contribution of diquat from the total optical density at 600 nm of the mixture.

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.

General formulas of the estimated likelihood ratio Y/X in the diagnosis of paternity of a deceased putative father

SummaryWhen the putative father is dead his probable genotype, essential for estimate of likelihood ratio in the diagnosis of paternity, should be deduced from his relatives. In the present paper are described the general method for such deduction of probable genotype and the derivation of the formula of likelihood ratio. 104 examples of the diagnosis of paternity of deceased father are examined using a Monte Carlo method and the distributions of relative frequencies of log(Y/X) are calculated for the true father and non-father. These results indicate that the present method of estimation of Y/X from relatives is quite useful for the diagnosis of paternity of a deceased putative father.ZusammenfassungWenn der vermutliche Vater verstorben ist, wird sein wahrscheinlicher Genotyp, der für eine Vaterschaftsbeurteilung notwendig ist, aus den Blutgruppenbefunden seiner Verwandten hergeleitet. In der vorgelegten Arbeit wird eine allgemeine Methode für eine solche Ableitung sowie die Herleitung einer Berechnungsformel des Likelihood-Quotienten Y/X dargelegt. 104 mit der Monte-Carlo-Methode hergestellte Fälle mit verstorbenem vermutlichem Vater wurden in dieser Weise ausgewertet und die Verteilung der relativen Häufigkeit von log (Y/X) für Väter und Nichtväter berechnet. Die Ergebnisse zeigen, daß die hier vorgestellte Methode zur Berechnung von Y/X aus Blutgruppenbefunden von Verwandten für die Vaterschaftsbeurteilung eines verstorbenen vermutlichen Vaters brauchbar ist.

Determination of molar absorptivities of radicals of 18 phenothiazine derivatives

Abstract A new colorimetric method is described for the quantification of 18 different phenothiazine derivatives. The phenothiazine derivatives changed immediately to various different colored radicals following their oxidation with 2,3-dichloro-5,6-dicyano- p -benzoquinone in 47% or 66% perchloric acid solution. The radicals produced were very stable. The extent of color fading after 1 h was less than 2% for all 18 radicals, and that after 24 h was less than 2% for 16 radicals. The absorption maxima of the 18 radicals in the visible region showed a linear concentration dependence in the range 0.5–10 μg/ml phenothiazine. The molar absorptivities of these radicals were determined in the ultraviolet and visible regions. With the exception of two derivatives studied, these radicals exhibited one prominent peak in the wavelength range 280–300 nm having a molar absorptivity ∼40 000 while the other peak occured between 500–640 nm having a molar absorptivity ∼10 000. These 18 phenothiazine derivatives were classified into seven groups according to the wavelength of the absorption maximum in the visible region.

Determination of paraquat in raw and formalin-fixed tissues by Electron Spin Resonance (ESR) spectroscopy

SummaryESR method was applied to determine paraquat levels in fresh and formalin-fixed tissues. Paraquat was converted to paraquat radical by adding sodium dithionite to tissue homogenates and detected by ESR. Paraquat levels of more than 0.2 μg/ml homogenate could be quantified with 0.1 ml of the homogenate. The use of manganese ions for standardization of paraquat signal enabled much more accurate ESR measurements because this ion was quite stable and its signal did not overlap that of paraquat. Even with tissues fixed in formalin, tissues paraquat levels were measurable after removing formalin from the tissue extract. This fact was verified by studying two cases; the tissues were kept in formalin for 1.5 years in case 1 and for 6.5 years in case 2. In both cases, the paraquat contents in tissues were 0.02–0.08 μg/g. In this way ESR is one of the most suitable methods in determining low levels of paraquat in tissues even after they were preserved in formalin for a long time.ZusammenfassungDas Elektronen-Spin-Resonanz-Verfahren (ESR) wurde zur Bestimmung von Paraquatkonzentrationen in frischen und formalinfixierten Geweben herangezogen. Die Bildung des Paraquat-Radikals aus Paraquat erfolgt mit Hilfe eines Zusatzes von Natriumdithionit. Mit dem beschriebenen ESR-Verfahren können Paraquatkonzentrationen von über 0.2 μg/ml beim Einsatz von 0.1 ml Homogenisat bestimmt werden. Durch die Verwendung von Manganionen als Eichsubstanzen bei der ESR-Messung konnte eine bessere Standardisierung erzielt werden. Dieses Ion ist hinsichtlich der Signale recht stabil und interferiert nicht mit dem Paraquat-Signalen. Selbst im Falle formalinfixierter Gewebeschnitte gelang die quantitative Bestimmung des Paraquat, wenn zuvor das Formalin entfernt wurde. Die Methode wurde auf zwei Fälle angewandt: Bei Fall 1 betrug die Formalinverweildauer 1,5 Jahre, bei Fall 2 6,5 Jahre. In beiden Fällen lagen die Paraquatkonzentrationen in den Geweben zwischen 0,02 und 0,08 μg/g. Es konnte gezeigt werden, daß die ESR-Methode außerordentlich geeignet ist, um selbst in formalinfixierten Geweben nach langer Expositionszeit auch niedrige Paraquatkonzentrationen zu bestimmen.

Rapid quantitative analysis of paraquat by electron spin resonance spectroscopy

A very simple and sensitive method for quantitative measurement of paraquat in plasma, urine and some drinks is described. The paraquat radical formed by its reduction can be detected by electron spin resonance spectroscopy (ESR) at room temperature without clean-up or concentration process of samples. The sensitivity limit is 0.1 microgram paraquat/ml and the required amount of sample is 100 microliter. The time needed for the measurement is within 10 min.

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.

Increase in production of ascorbate radical in tissues of rat treated with paraquat

The production of ascorbate radical (A·-) was investigated in tissues of rats intoxicated with paraquat (PQ) to know the protective role of antioxidant ascorbate (AH·-) in tissues. The electron spin resonance (ESR) method is applied to observe A·-. To eliminate increased biosynthesis of ascorbic acid (AH2) by PQ intoxication, ODS rats were chosen and fed with or without 250 ppm PQ in the diet. The radical A·- was detected only in the lung and spleen homogenates of both intoxicated and control rats at the beginning of ESR measurement. The radical levels of intoxicated rat lung and spleen were increased rapidly to twice the initial level after 3 h and decreased to 0.2–0.6 times the initial level after 24 h, whereas those of control rats were increased slowly to 1.1 times the initial level after 4 h and decreased slowly to 0.7 times the initial level after 24 h at 4°C. In other organs such as liver, kidney, heart and testis, A·- was not detected initially but detected afterwards. Higher A·- level was observed in the intoxicated rat liver than the control but no appreciable differences of A·- levels were observed between the intoxicated kidney, heart and testis and the respective controls. In the intoxicated rat lung the concentration of AH2 is only half but that of A·- is twice as high as that of the control. Larger amounts of A·- produced in the intoxicated rats decayed more quickly than those in the control rats. The simple addition of PQ to the control organ enhanced neither A·- production nor A·- quenching. These facts suggest that the tissues damaged by PQ require larger amounts of AH- to detoxicate harmful oxidants, resulting in concomitant production of A·-.