A highly sensitive quantification method for 12 plant toxins in human serum using liquid chromatography tandem mass spectrometry with a quick solid-phase extraction technique.

We developed a highly sensitive quantification method using liquid chromatography tandem mass spectrometry (LC/MS/MS) for 12 plant toxins in human serum. In this paper, we selected lycorine, galanthamine, protoveratrine A, protoveratrine B, veratramine, veratridine, jervine, cyclopamine, cevadine, α-solanine, α-chaconine, and solanidine as targeted analytes. The ADME column was utilized for LC separation and a Monolithic SPE column (MonoSpin® C18) for analyte extraction. The total time for SPE clean-up and LC/MS/MS analysis was completed within 30 min. The method validation results were as follows: the linearity (r2) of each calibration curve was over 0.99; the inter- and intra-day accuracies were 92.7 %-116 % and 91.6 %-106 %, respectively; and the inter- and intra-day precisions were below 14 % and 11 %, respectively. Also, the lower limits of detection and quantification were 0.0071-0.15 and 0.022-0.46 ng/mL, respectively, indicating the method's high sensitivity. Finally, to confirm its feasibility, our method was applied to two model samples: (1) commercially available human serum and (2) pseudo poisoning serum via dilution of mouse serum with human serum. We were able to quantify α-chaconine at 0.84 ± 0.02 ng/mL in the serum (Case 1) and protoveratrine A at 0.15 ± 0.032 ng/mL in the pseudo poisoning serum (Case 2), demonstrating our method's practicality. This is the first time that the 12 plant toxins in human serum were simultaneously quantitated. Our method can investigate accidental poisonings involving toxic plants, enabling prompt decisions on patient treatment.

Quantitative analysis of the Tricholoma ustale-derived toxin, ustalic acid, in mushroom and food samples by LC-MS/MS.

Tricholoma ustale, a poisonous member of the Tricholomataceae family, causes gastrointestinal symptoms such as diarrhea and vomiting. In Japan, 86 cases (affecting a total of 347 patients) of poisoning with Tricholoma ustale have been reported between 1989 and 2010. Ustalic acid is one of the primary toxic components in Tricholoma ustale. In the present study, the quantitative analysis of the ustalic acid content in mushroom and food samples was conducted by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Mushroom and food samples were extracted using methanol containing 0.5% formic acid and 50% aqueous methanol, respectively. Purification using SAX solid-phase extraction (SPE) was conducted prior to LC-MS/MS analysis, which was performed in the ESI negative mode using a C18 column. The method developed for the LC-MS/MS analysis of ustalic acid was extremely sensitive. The limits of quantitation calculated at a signal-to-noise ratio of 10 were 10ng/g (shiitake mushroom) and 0.40ng/g (miso soup). The accuracies of quantitation in the shiitake mushroom and miso soup samples ranged from 99.8%-105% and 98.8%-102%, respectively. This method was applied to leftover mushroom samples from a food poisoning case; here, ustalic acid was detected at 0.57, 3.7µg/g. This analytical method using LC-MS/MS could be useful in food poisoning cases involving mushrooms. This is the first report in which the ustalic acid content was determined using the leftovers of a food poisoning case.

Determination of Residues of Phenicol Drugs in Ayu (Plecoglossus altivelis) by LC-MS/MS.

An analytical method for the determination of residues of 3 phenicol drugs (chloramphenicol, thiamphenicol and florfenicol) in Ayu (Plecoglossus altivelis) by LC-MS/MS was developed. We used the whole body of Ayu, including the bones and internal organs, in addition to muscle. Phenicols were extracted with 90% acetonitrile and an aliquot of the crude extract was cleaned up on a Florisil column (2 g), followed by defatting with n-hexane. The acetonitrile extract was evaporated and the solvent was replaced with phosphate buffer, then the extract was purified on a hydroxylated styrene-divinylbenzene copolymer column (200 mg). Finally, sample solution was passed through a deproteination cartridge filter with a lipid removal function. Chloramphenicol was quantitated by means of a calibration curve corrected with salogate standard (chloramphenicol-d5) and thiamphenicol and florfenicol were quantitated based on absolute calibration curves. This method was validated in accordance with the notification of the Ministry of Health, Labour and Welfare of Japan. As a result of the validation study, the trueness, repeatability and within-laboratory reproducibility were 85-103, 5-13 and 8-13%, respectively. This method is useful for inspecting residues of 3 phenicol drugs in whole body of Ayu efficiently. Moreover, when chloramphenicol and thiamphenicol are detected by this method, the quantitated value is applicable to decide the compliance of the sample with the specifications and standards of the Food Sanitation Law.

[Determination of Butroxydim in Agricultural Products by LC-MS].

An analytical method for the determination of butroxydim in agricultural products by LC-MS was developed. Butroxydim was extracted with acetonitrile and an aliquot of the crude extract was cleaned up on an octadecyl silanized silica gel (C18) cartridge column (1,000 mg), followed by a salting-out step to remove water. Before purification on a silica gel (SI) cartridge column (690 mg), polar matrices were precipitated by adding ethyl acetate, n-hexane and anhydrous sodium sulfate successively. This process effectively removed caffeine and catechins and improved recovery when analyzing residual butroxydim in tea leaves. Recovery and repeatability were good; the relative standard deviations were less than 5% for all 12 tested agricultural products (brown rice, soybean, potato, spinach, cabbage, apple, orange, grapefruit, lemon, tomato, peas with pods, and tea). Average recoveries for 11 agricultural products, except for lemon, were 74-92%.

Development of a Method for Crustacean Allergens Using Liquid Chromatography/Tandem Mass Spectrometry.

An LC/MS/MS analysis method was developed for crustacean allergens, tropomyosin, and arginine kinase. A protein extract from shrimp was reduced, alkylated, and digested by trypsin. Peptide spectra were obtained using full scan analysis by LC/MS/MS, and we determined a sequence through a protein search. 22ADTLEQQNK30, 92IQLLEEDLER101, 113LAEASQAADESER125, 134SLSDEER140, 153FLAEEADR160, and 190IVELEEELR198 of tropomyosin and 152VSSTLSSLEGELK164 and 217TFLVWVNEEDHLR229 of arginine kinase were selected as the specific peptides, and optimal multiple-reaction monitoring conditions were used. The results obtained through the LC/MS/MS analysis correlated well with those using the ELISA method for various crustacean samples (r2>0.9). Moreover, unregulated species, such as krill or insects, which produce positive results in some crustacean ELISA assays, can be differentiated by LC/MS/MS. These findings suggest that LC/MS/MS analysis may be effective for crustacean food allergen analysis.

[Radioactive cesium analysis in radiation-tainted beef by gamma-ray spectrometry with germanium semiconductor detector].

The detection limit and precision of radioactive cesium measurement in beef by gamma-ray spectrometry with a germanium semiconductor detector were evaluated. Measurement for 2,000 seconds using a U-8 container (100 mL) provided a detection limit of radioactive cesium (the sum of 134Cs and 137Cs) of around 20 Bq/kg. The 99% confidence interval of the measurement of provisional maximum residue limit level (491 Bq/kg) samples ranged from 447 to 535 Bq/kg. Beef is heterogeneous, containing muscle and complex fat layers. Depending on the sampled parts, the measurement value is variable. It was found that radioactive cesium content of the muscle layer was clearly different from that of fat, and slight differences were observed among parts of the sample (SD=16.9 Bq/kg), even though the same region (neck block) of beef sample was analyzed.

Pyrene-sensitized electron transport across vesicle bilayers: Dependence of transport efficiency on pyrene substituents.

Endoergic electron transport across vesicle bilayers from ascorbate (Asc-) in the inner waterpool to methylviologen (MV2+) in the outer aqueous solution was driven by the irradiation of pyrene derivatives embedded in the vesicle bilayers. The initial rate of MV2+ reduction is dependent on the substituent group of the pyrenyl ring; a hydrophilic functional group linked with the pyrenyl ring by a short methylene chain acts as a sensitizer for the electron transport. Mechanistic studies using (1-pyrenyl)alkanoic acids (1a-c) as sensitizers suggest that the electron transport is mainly initiated by the reductive quenching of the singlet excited state of the pyrene by Asc- and proceeds by a mechanism involving electron exchange between the pyrenes located at the inner and outer interface across the vesicle bilayer. We designed and synthesized novel unsymmetrically substituted pyrenes having both a hydrophilic group linked by a short methylene chain and a hydrophobic long alkyl group (5a-c), which acted as excellent sensitizers for the electron transport across vesicle bilayers.