Development of a high-performance liquid chromatography-tandem mass spectrometric method for the determination of Methimazole in human blood matrices.

Methimazole (MMI, 1-methyl-2-mercaptoimidazole) is widely used for the treatment of hyperthyroidisms. There are methods available for the measurement of MMI concentration in human serum or plasma from the past, but none meet the current regulatory standards for bioanalytical method validations. In this paper, we developed and validated a total MMI measurement method using liquid chromatography-tandem mass spectrometry (LC-MS/MS), a technique that conforms to current bioanalytical method validation. To form a free sulfhydryl group on MMI, sodium bisulfite was added to 50 µl of plasma or serum samples containing MMI before the derivatization step. The internal standard (MMI-D3) was spiked into samples, then these samples were derivatized with 4-chloro-7-nitro-2,1,3-benzoxadiazole. After derivatization, these samples were extracted by supported liquid extraction. Then, the organic solvent was evaporated and the residue was dissolved in 50% methanol and injected into the LC-MS/MS system. A calibration curve was plotted over the concentration range 1-1000 ng/mL (r2 = 0.999). The intra-day and inter-day precisions were less than 10.2% and 9.8%, respectively. The intra-day and inter-day accuracies were between 89.5% and 101.1%, and 96.0% and 99.7%, respectively. The long-term stability of samples showed good precision and accuracy. The validated method was successfully applied to determine serum total MMI concentration in Graves' disease patients after oral administration of 5, 10 or 15 mg MMI. The range of circulating total MMI concentrations was found to be between 2.69 and 304.27 ng/mL in this study. It was shown that the measured serum total MMI concentrations changed in a dose-dependent manner.

Probabilistic alignment leads to improved accuracy and read coverage for bisulfite sequencing data.

BACKGROUND: DNA methylation has been linked to many important biological phenomena. Researchers have recently begun to sequence bisulfite treated DNA to determine its pattern of methylation. However, sequencing reads from bisulfite-converted DNA can vary significantly from the reference genome because of incomplete bisulfite conversion, genome variation, sequencing errors, and poor quality bases. Therefore, it is often difficult to align reads to the correct locations in the reference genome. Furthermore, bisulfite sequencing experiments have the additional complexity of having to estimate the DNA methylation levels within the sample. RESULTS: Here, we present a highly accurate probabilistic algorithm, which is an extension of the Genomic Next-generation Universal MAPper to accommodate bisulfite sequencing data (GNUMAP-bs), that addresses the computational problems associated with aligning bisulfite sequencing data to a reference genome. GNUMAP-bs integrates uncertainty from read and mapping qualities to help resolve the difference between poor quality bases and the ambiguity inherent in bisulfite conversion. We tested GNUMAP-bs and other commonly-used bisulfite alignment methods using both simulated and real bisulfite reads and found that GNUMAP-bs and other dynamic programming methods were more accurate than the more heuristic methods. CONCLUSIONS: The GNUMAP-bs aligner is a highly accurate alignment approach for processing the data from bisulfite sequencing experiments. The GNUMAP-bs algorithm is freely available for download at: http://dna.cs.byu.edu/gnumap. The software runs on multiple threads and multiple processors to increase the alignment speed.

Investigation on the presence of sulphites in fresh meat preparations: estimation of an allowable maximum limit.

Sulphiting agents are commonly used food additives. They are not allowed in fresh meat preparations. In this work, 2250 fresh meat samples were analysed to establish the maximum concentration of sulphites that can be considered as "natural" and therefore be admitted in fresh meat preparations. The analyses were carried out by an optimised Monier-Williams Method and the positive samples confirmed by ion chromatography. Sulphite concentrations higher than the screening method LOQ (10.0 mg · kg(-1)) were found in 100 samples. Concentrations higher than 76.6 mg · kg(-1), attributable to sulphiting agent addition, were registered in 40 samples. Concentrations lower than 41.3 mg · kg(-1) were registered in 60 samples. Taking into account the distribution of sulphite concentrations obtained, it is plausible to estimate a maximum allowable limit of 40.0 mg · kg(-1) (expressed as SO(2)). Below this value the samples can be considered as "compliant".

Levels of sulfite in three organs from mice exposed to sulfur [corrected] dioxide.

To study whether sulfur dioxide (SO(2)) can enter into the different organs of mice exposed to SO(2), the sulfite contents in brains, hearts, and lungs from male mice were determined by high-performance liquid chromatography with fluorescence detection (HPLC-FD). After reduction and precolumn derivation of tissue homogenates of brains, hearts, and lungs from mice, the mixture was centrifuged, and 5 microl of the resulting supernatant was directly injected into HPLC; the mobile phase consisted of methanol-phosphoric acid (12:88, v/v), and for the fluorescence detection lambdaEX 392 nm and lambdaEM--479 nm were used. The standard curve was linear in the range from 0.126 microg/ml to 126 microg/ml; the minimal detectable concentration was 0.04 microg/ml (S/N = 3), the average methodological recoveries were from 97% to 101%, and the within-day and between-day precisions were less than 9%. These results showed that sulfite contents in all organs tested from mice in the SO(2)-exposed groups were significantly increased (p < .05) in a dose-dependent manner (r > .92) compared with the control groups. These results indicated SO(2) could transform into sulfite in vivo after inhalation, and could distribute into lung and other organs such as brain and heart. These results offered a support for the viewpoint that SO(2) is a systemic toxic agent.

Utilization of the skin attachment model to determine the antibacterial efficacy of potential carcass treatments.

Two experiments (EXP), utilizing the skin attachment model (SAM), were conducted to determine the bactericidal activity of six potential carcass disinfectants [EXP 1: 20, 400, and 800 ppm sodium hypochlorite; EXP 2: 5% acetic acid (AA), 8% trisodium phosphate (TSP), and 1% sodium metabisulfite (SS)] during simulated scalder (50 C for 2 min), chiller (0 C for 60 min), or post-process dip (23 C for 15 s) application. Efficacies of treatments were determined against populations of Salmonella typhimurium that were "loosely" or "firmly" attached to chicken breast skin (10 cm diameter). For comparison, activity of the six disinfectants was also determined against S. typhimurium in aqueous suspension. All disinfectants except SS reduced numbers of freely suspended S. typhimurium by > or = 4.5 log10 cfu/mL. The sodium metabisulfite did not reduce populations of salmonellae. In both EXP, there were disinfectant by application interactions (P < 0.05) on activity against loosely and firmly attached cells. Sodium hypochlorite at 20 ppm had little activity regardless of application, whereas higher levels were more effective (P < 0.001), particularly in the chiller application, in which loosely and firmly attached populations were reduced by 2.3 to 2.5 and 1.3 to 1.9 log10 cfu per skin, respectively. In EXP 2, SS showed no activity regardless of application. Trisodium phosphate was similarly effective (reduction by 1.2 to 1.8 log10 cfu per skin) in all applications (P > 0.05). In contrast, AA activity was affected by the application method (P < 0.05). Against loosely attached cells, AA was most effective in the chiller application (2.5 log10 reduction), whereas against firmly attached cells, AA was effective only in the scalder application (2.0 log10 reduction). Attachment of S. typhimurium to poultry skin apparently increased the ability of the bacteria to resist various disinfectants, and efficacy was influenced by extent of attachment of bacteria to skin and method of disinfectant application.