Yoshie Hayashizaki

Rapid drug extraction from human whole blood using a modified QuEChERS extraction method

A modified QuEChERS (quick, easy, cheap, effective, rugged and safe) extraction method followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) has been developed for the simultaneous determination of forensically important drugs and poisons (more than 90 compounds) in human whole blood. Because the QuEChERS method is commonly used for the analysis of pesticide residues in foods, we customized the QuEChERS method for forensic use. This extraction method consists essentially of two steps: (1) extraction/partitioning and (2) dispersive-solid phase extraction. In step 1, three-fold diluted blood was mixed with an internal standard (D5-diazepam for basic drugs or D5-phenobarbital for acidic drugs) solution, a QuEChERS pre-packed extraction kit (containing magnesium sulfate and sodium acetate) and a stainless steel bead, then partitioned into three layers by centrifugation. In step 2, the top layer (acetonitrile) was transferred into a centrifuge tube containing a dispersive-solid phase sorbent (containing primary secondary amine, end-capped octadecylsilane, and magnesium sulfate) and mixed for purification. After the centrifugation, supernatant was injected into LC-MS/MS. The QuEChERS method was applied in an autopsy case and we confirmed that this method can easily extract various types of drugs and metabolites from human whole blood. The combination of the modified QuEChERS method and LC-MS/MS could enable technicians inexperienced in forensic toxicological analysis to acquire reliable data quickly and easily.

Rapid determination of disulfoton and its oxidative metabolites in human whole blood and urine using QuEChERS extraction and liquid chromatography–tandem mass spectrometry

A liquid chromatography-tandem mass spectrometry method was developed and validated for simultaneous determination of disulfoton and five of its oxidative metabolites (disulfoton-sulfoxide, disulfoton-sulfone, demeton-S, demeton-S-sulfoxide and demeton-S-sulfone) in human whole blood and urine. Extraction was undertaken using a QuEChERS method, which is commonly used in food analysis. D10-Disulfoton was used as the internal standard. Separation was carried out using a CAPCELL-PAK MG II column (35×2.0 mm i.d., 5 μm, Shiseido) with a mobile phase of 10 m mol/L ammonium formate and methanol. This method was applied in an autopsy case, and disulfoton and its oxidative metabolites were successfully detected in both blood and urine. The concentrations of disulfoton in the blood and urine were 360 and 23.8 ng/mL, respectively. There was a relatively low concentration of demeton-S in both the blood (4.0 ng/mL) and urine (45.7 ng/mL). To date, there have been no reported cases of detection of demeton-S in human samples.

Diagnosis of drowning using post-mortem computed tomography based on the volume and density of fluid accumulation in the maxillary and sphenoid sinuses.

Recent studies have reported that drowning victims frequently have fluid accumulation in the paranasal sinuses, most notably the maxillary and sphenoid sinuses. However, in our previous study, many non-drowning victims also had fluid accumulation in the sinuses. Therefore, we evaluated the qualitative difference in fluid accumulation between drowning and non-drowning cases in the present study. Thirty-eight drowning and 73 non-drowning cases were investigated retrospectively. The fluid volume and density of each case were calculated using a DICOM workstation. The drowning cases were compared with the non-drowning cases using the Mann-Whitney U-test because the data showed non-normal distribution. The median fluid volume was 1.82 (range 0.02-11.7) ml in the drowning cases and 0.49 (0.03-8.7) ml in the non-drowning cases, and the median fluid density was 22 (-14 to 66) and 39 (-65 to 77) HU, respectively. Both volume and density differed significantly between the drowning and non-drowning cases (p=0.001, p=0.0007). Regarding cut-off levels in the ROC analysis, the points on the ROC curve closest (0, 1) were 1.03ml (sensitivity 68%, specificity 68%, PPV 53%, NPV 81%) and 27.5 HU (61%, 70%, 51%, 77%). The Youden indices were 1.03ml and 37.8 HU (84%, 51%, 47%, 86%). When the cut-off level was set at 1.03ml and 27.5HU, the sensitivity was 42%, specificity 45%, PPV 29% and NPV 60%. When the cut-off level was set at 1.03ml and 37.8HU, sensitivity was 58%, specificity 32%, PPV 31% and NPV 59%.

Simultaneous determination of 11 aconitum alkaloids in human serum and urine using liquid chromatography–tandem mass spectrometry

A liquid chromatography-tandem mass spectrometry method was developed and validated for simultaneous determination of four aconitines (aconitine, mesaconitine, hypaconitine, jesaconitine) and seven of their hydrolysis products (benzoylmesaconine, benzoylhypaconine, 14-O-anisoylaconine, benzoylaconine, aconine, mesaconine, hypaconine) in human serum and urine samples. Extraction was undertaken using a mixed-mode cation-exchange cartridge (OASIS MCX), and D(5)-aconitine was used as an internal standard. Separation of aconitum alkaloids was carried out using an L-column ODS with the mobile phase consisting of 10mM ammonium formate and methanol. The intra- and inter-day precisions were 0.3% to 9.9% and 3.2% to 12.8%, respectively. Intra- and inter-day accuracies were -14.1% to 7.3%, and -10.6% to 8.3%, respectively. The limit of detection and limit of quantification of analytes were 0.04-0.38 ng/mL and 0.12-1.15 ng/mL respectively. This method was applied in an autopsy case and successfully detected aconitines and their metabolites as well as some anti-psychiatric drugs.

Assessment of the relationship between drowning and fluid accumulation in the paranasal sinuses on post-mortem computed tomography.

Recent reports have detailed common computed tomography (CT) findings in drowning victims, most notably fluid accumulation in the maxillary and sphenoidal sinuses. This CT finding could help forensic doctors to diagnose drowning. This study retrospectively investigated 151 subjects: 39 drowning and 112 non-drowning cases. Pearsons chi-square tests demonstrated that fluid accumulation in the maxillary or sphenoidal sinuses was associated significantly with drowning (p=0.0001). The sensitivity of the drowning diagnosis was 97%, specificity was 35%, accuracy was 51%, positive predictive value was 34% and negative predictive value was 98%. Drowning was significantly associated with fluid accumulation in the maxillary and sphenoidal sinuses, but the specificity and positive predictive value of the drowning diagnosis were poor. Although the presence of fluid in the maxillary and sphenoidal sinuses cannot be used to diagnose drowning, the absence of the fluid can be used to virtually exclude drowning.

Quantitative analysis of 3,4-dimethylmethcathinone in blood and urine by liquid chromatography–tandem mass spectrometry in a fatal case

We report here the quantitative analysis of cathinone-type designer drug 3,4-dimethylmethcathinone (3,4-DMMC) in blood and urine using liquid chromatography-tandem mass spectrometry (LC-MS/MS) in a fatal case. Abuse of 3,4-DMMC is widespread and a global issue. However, to date, there have been no reports of 3,4-DMMC-related deaths. We encountered a death in which 3,4-DMMC was thought to play a causative role, and successfully identified this designer drug from biological samples by using LC-MS/MS and QuEChERS (quick, easy, cheap, effective, rugged and safe) extraction method. For standard samples, detection of 3,4-DMMC in human blood and urine samples in the calibration range (5-400 ng/mL) was successful with recoveries of 85.9-89.4% (blood) and 95.8-101% (urine), limits of detection of 1.03 (blood) and 1.37 ng/mL (urine) and limits of quantification of 5.00 (blood) and 5.38 ng/mL (urine). The concentrations of 3,4-DMMC in blood (external iliac vein) and urine in the case were 27 mg/L and 7.6 mg/L, respectively. Some metabolites, including 3,4-dimethylcathione (DMC) and β-ketone reduced metabolites (β-OH-DMMC and β-OH-DMC), were detected in both blood and urine.

Hypothermic death: Possibility of diagnosis by post-mortem computed tomography

Referring to our experience with post-mortem computed tomography (CT), many hypothermic death cases presented a lack of increase in lung-field concentration, blood clotting in the heart, thoracic aorta or pulmonary artery, and urine retention in the bladder. Thus we evaluated the diagnostic performance of post-mortem CT on hypothermic death based on the above-mentioned three findings. Twenty-four hypothermic death subjects and 53 non-hypothermic death subjects were examined. Two radiologists assessed the presence or lack of an increase in lung-field concentration, blood clotting in the heart, thoracic aorta or pulmonary artery, and measured urine volume in the bladder. Pearsons chi-square test and Mann-Whitney U-test were used to assess the relationship between the three findings and hypothermic death. The sensitivity, specificity, accuracy, positive predictive value (PPV) and negative predictive value (NPV) of the diagnosis were also calculated. Lack of an increase in lung-field concentration and blood clotting in the heart, thoracic aorta or pulmonary artery were significantly associated with hypothermic death (p=0.0007, p<0.0001, respectively). The hypothermic death cases had significantly more urine in the bladder than the non-hypothermic death cases (p=0.0011). Regarding the diagnostic performance with all three findings, the sensitivity was 29.2% but the specificity was 100%. These three findings were more common in hypothermic death cases. Although the sensitivity was low, these findings will assist forensic physicians in diagnosing hypothermic death since the specificity was high.

Sex determination of the pelvis using Fourier analysis of postmortem CT images

BACKGROUND The Fourier transform (FT) is an image-processing tool that can be used to analyze space or linear systems. We used the FT to analyze the shape of the subpubic line (SL) and the right greater sciatic notch area line (GSNAL) of the pelvis to identify sexing in computed tomographic (CT) images. METHODS 120 bodies (60 male, 60 female) were scanned by multislice CT before autopsy. SL and GSNAL images were reconstructed using an image workstation. The range of the SL is between the bottom ends of the bilateral acetabulum, and the range of the right GSNAL is from the posterior superior iliac spine to the ischial tuberosity. Coordinates (x, y) of the bone outline were extracted from CT images and calculated by fast FT. The Mann-Whitney U test comparing the amplitude of each frequency was used to determine significant differences between the two populations. Multiple regression analyses were performed to find statistically significant amplitudes of each frequency. We verified the accuracy of prediction of the regression equation, and a Cohens kappa coefficient was measured. RESULTS Statistically significant differences of amplitude were found in the 2nd, 3rd, and 4th frequencies of the SL and the 1st, 3rd, and 4th frequencies of GSNAL. Adjusted R(2) using all amplitudes having a significant difference was 0.772. The accuracy was 0.983. Kappa coefficient was 0.983. CONCLUSION We could determine sexing from SL and GSNAL of the pelvis using FT with high accuracy. FT is a useful tool to analyze the curved shape of human bone.

Radiological analysis of a naturally mummified body

A body in an advanced stage of mummification was found in a concrete apartment in Japan. Natural complete mummification is very rare in Japan’s humid and temperate climate. We performed multi-slice computed tomography (MSCT) to obtain information on the body prior to autopsy. MSCT clearly illustrated old fractures in the right lower leg. Bone resists destruction during body decomposition. If antemortem medical records of the deceased are available, MSCT scanning can provide information for positive identification. At autopsy, the cervical tissues presented as a dried mass, and it was difficult to separately remove the hyoid bone and thyroid cartilage. Fractures of the large horn of the hyoid bone and superior horn of the thyroid cartilage, which are not observed in all strangulation cases, strongly suggest criminal activity. The diagnosis of these fractures is of great value. In our case, MSCT revealed that there was no fracture in the hyoid bone or ossified area of the thyroid cartilage. Hard tissues are usually well preserved in mummies. Although MSCT images have limits in mummies because of the severe dryness of soft tissues and organs, they could become a useful tool not only for personal identification, but also for the identification of neck compression.

Usefulness of postmortem computed tomography before forensic autopsy for alerting forensic personnel to tuberculosis infection

Since May 2009, we have performed multislice computed tomography (MSCT) prior to forensic autopsy for cases of suspicious death. In the present case, innumerable widely scattered nodules in both pulmonary fields on MSCT were indicative of miliary tuberculosis (TB). At autopsy, both lungs were submerged in formalin fluid immediately after removal from the body. Miliary TB was finally diagnosed based on microscopic findings. TB is a disease that autopsy room workers need to be aware of to protect themselves. Unfortunately, because little medical information about deceased individuals is usually available before forensic autopsy, the diagnosis of TB is frequently not made until autopsy. This leads to a much higher incidence of TB in autopsy room staff members even if they wear protective clothing. Therefore, MSCT before forensic autopsy may identify suspected cases of miliary TB in advance and thus help to prevent TB infection in forensic autopsy personnel.