Proposal for risk-based scientific approach on full and partial validation for general changes in bioanalytical method.

The guidance and several guidelines on bioanalytical method validation, which were issued by the US FDA, EMA and Ministry of Health, Labour and Welfare, list the 'full' validation parameters; however, none of these provide any details for 'partial' validation. Japan Bioanalysis Forum approved a total of three annual discussion groups from 2012 to 2014. In the discussion groups, members from pharmaceutical companies and contract research organizations discussed the details of partial validation from a risk assessment viewpoint based on surveys focusing on bioanalysis of small molecules using LC-MS/MS in Japan. This manuscript presents perspectives and recommendations for most conceivable changes that can be made to full and partial validations by members of the discussion groups based on their experiences and discussions at the Japan Bioanalysis Forum Symposium.

Identification and some properties of anthocyanin isolated from Zuiki, stalk of Colocasia esculenta.

Zuiki, a stalk of taro (Colocasia esculenta), is a traditional vegetable in Japan. Raw zuiki is often boiled and vinegared to eat. The surface color of zuiki is reddish. Here, we isolated a red pigment from zuiki and identified it as cyanidin 3-rutinoside using instrumental analyses. The color of zuiki disappeared by boiling, but the zuiki turned red again in an acetic acid solution. It seems that the cyanidin 3-rutinoside that exists on the surface of zuiki elutes in boiling water and then, the pigment that seeps out from the inside of the zuiki is exposed to an acid solution, and its surface turns red again. The radical scavenging activity of purified zuiki anthocyanin was 114 mg equivalent to BHT/g. About half of the anthocyanin in fresh zuiki was washed out by boiling, and the radical scavenging activity of zuiki was definitely reduced.

Optimization of a method for determining dioxin in whole blood samples based on solvent extraction and simplified cleanup.

Dioxins, including polychlorinated dibenzo-p-dioxins/dibenzofurans (PCDDs/Fs) and coplanar polychlorinated biphenyls (Co-PCBs), such as mono-ortho-PCBs and non-ortho-PCBs, are environmental pollutants that have deleterious effects on human health. Although screening of blood samples for dioxins is necessary, the current methods are time-, reagent- and labor-intensive. To optimize the extraction and cleanup of dioxins, we have designed a column chromatography method, coupled with a water washing step. We used a tandem simplified multilayer silica gel-activated carbon dispersed silica gel column (TS-ML-AC) rather than the conventional two columns. We compared three liquid-liquid extraction (LLE) methods and two pressurized liquid extraction (PLE) methods, when used with this column. For each of these extraction methods, we compared the quantity of lipid obtained when the water washing step was omitted and when it was performed by shaking 30 times by hand or 30 min by a machine. We found that TS-ML-AC was superior to the conventional pair of columns in that only about one third of the solvent and only one quarter of the time was necessary. Of the five extraction methods, the acetone/hexane PLE (AcP) method was superior, since it reduced the amount of organic solvent to half or less of the amount required for the LLE methods. The cleanup step using water was best accomplished by the hand-shaking method. Our results indicate that, for the analysis of dioxin in whole blood samples, the use of AcP together with TS-ML-AC and water washing by hand shaking should be used.

Optimization of the purification method for dioxin analysis in human serum and temporal changes in background dioxin levels in the general population.

To perform high quality and high throughput measurements, complicated purification procedure by two rounds of open column chromatography was simplified by appraising the components and adding a sufficient amount of adsorbents to the column. The procedure consists of two open column techniques using a multi-layer silica gel column and an active carbon dispersed silica gel column. This purification procedure is used in "Standard manual for dioxin analysis in human blood" published in 2000 (Ministry of Health and Welfare, Japan, 2000). Consequently, a purification procedure using only 1 g of 10% AgNO(3)/silica gel can remove the matrix of blood serum as effectively as a multi-layer silica gel column. In addition, 0.1 g of carbon silica gel, equivalent to 1/10 the weight used currently in analyses, separates and recovers the dioxins in purified extract of blood serum. Furthermore, changes in the background level in the general population were clarified by monitoring the dioxin concentrations in human blood. The dioxin concentrations and ratios in blood in the general population remained at a tolerable level for three months. Therefore, the blood serum was available for the detection of a remarkable elevation in dioxins level considered to be indicative of contamination due to high-density exposure.