Determination of semicarbazide residue in human urine samples using liquid chromatography-tandem mass spectrometry.

An ultra-performance liquid chromatography coupled with electrospray ionisation tandem mass spectrometry (UPLC-ESI-MS/MS) with pre-column derivatisation was developed and validated for the determination of semicarbazide in human urine. Urine samples were derivatised with 2-nitrobenzaldehyde and subsequently extracted with acetonitrile. Extracts were concentrated and then analysed by UPLC-MS/MS. The time per run was 7 min. Good results were observed for the linearity of matrix-matched calibration curves (R2 > 0.99) in the concentration range of 1-100 µg/L. The absolute recovery ranged from 98.7% to 108.6%, with the relative standard deviations (RSDs) of 2.2%-3.6%. The limit of detection and quantification for the semicarbazide was 0.5 µg/L and 1 µg/L, respectively. The method showed good extraction efficiency, high sensitivity, and good reproducibility. It was suitable for the detection of semicarbazide in human urine. Residues of semicarbazide were between 1.0 and 41.5 µg/L in children's 24-h urine. This work is the first report on the quantitative analysis of SEM in 24-h human urine samples.

Chemoproteomic identification of molecular targets of antifungal prototypes, thiosemicarbazide and a camphene derivative of thiosemicarbazide, in Paracoccidioides brasiliensis.

Paracoccidioidomycosis (PCM) is a neglected human systemic disease caused by species of the genus Paracoccidioides. The disease attacks the host's lungs and may disseminate to many other organs. Treatment involves amphotericin B, sulfadiazine, trimethoprim-sulfamethoxazole, itraconazole, ketoconazole, or fluconazole. The treatment duration is usually long, from 6 months to 2 years, and many adverse effects may occur in relation to the treatment; co-morbidities and poor treatment adherence have been noted. Therefore, the discovery of more effective and less toxic drugs is needed. Thiosemicarbazide (TSC) and a camphene derivative of thiosemicarbazide (TSC-C) were able to inhibit P. brasiliensis growth at a low dosage and were not toxic to fibroblast cells. In order to investigate the mode of action of those compounds, we used a chemoproteomic approach to determine which fungal proteins were bound to each of these compounds. The compounds were able to inhibit the activities of the enzyme formamidase and interfered in P. brasiliensis dimorphism. In comparison with the transcriptomic and proteomic data previously obtained by our group, we determined that TSC and TSC-C were multitarget compounds that exerted effects on the electron-transport chain and cell cycle regulation, increased ROS formation, inhibited proteasomes and peptidases, modulated glycolysis, lipid, protein and carbohydrate metabolisms, and caused suppressed the mycelium to yeast transition.

Lipophilicity Studies on Thiosemicarbazide Derivatives.

The lipophilicity of two series of thiosemicarbazide derivatives was assessed by the RP-HPLC method with the RP-18 chromatographic column and the methanol-water mixture as the mobile phase. Distribution coefficients logPHPLC were compared to calculated values generated by commonly used AClogP software and quantum chemical calculations. The reliability of the predictions was evaluated using the correlation matrix and PCA. For 4-benzoylthiosemicarbazides, a high correlation between theoretical and experimental logP parameters was obtained using the XlogP3 algorithm, while for 4-aryl/(cyclohexyl)thiosemicarbazides, the XlogP2 parameter was strongly correlated with the experimentally obtained logP.

Rapid detection of economic adulterants in fresh milk by liquid chromatography-tandem mass spectrometry.

A method to aid in the detection of the economically driven adulteration of fresh milk with a range of small, nitrogen containing compounds, including melamine, ammeline, ammelide, cyanuric acid, allantoin, thiourea, urea, biuret, triuret, semicarbazide, aminotriazine, 3- and 4-aminotriazole, cyanamide, dicyandiamide, guanidine, choline, hydroxyproline, nitrate, and a range of amino acids, has been developed. (15)N2-Urea is used as an internal standard. The adulteration of milk with exogenous urea has previously been difficult to detect because of the variation in the naturally occurring levels of urea in milk. However, by monitoring the contaminants biuret and triuret, which comprise up to 1% of synthetic urea, the adulteration of milk with urea-based fertilizer can be detected. We estimate that to be economically viable, adulteration of the order of 90-4000ppm of the above adulterants would need to be added to fresh milk. For most of the compounds, an arbitrary detection threshold of 2ppm is therefore more than sufficient. For biuret, a lower detection threshold, better than 0.5ppm, is desirable and the sensitivity for biuret and triuret can be improved by the post-column addition of lithium to create lithium adducts under electrospray ionisation. Sample handling involves a two-step solvent precipitation method that is deployed in a 96-well plate format, and the hydrophilic interaction liquid chromatography uses a rapid gradient (1.2min). Three separate injections, to detect the positively charged compounds, the negatively charged compounds and amino acids and finally the lithium adducts, are used. This rapid and qualitative survey method may be deployed as a second tier screening method to quickly reduce sample numbers indicated as irregular by an FTIR based screening system, and to direct analysis to appropriate quantification methods.

Determination of semicarbazide in baby food by liquid chromatography/tandem mass spectrometry: interlaboratory validation study.

An interlaboratory validation study funded by the European Commission, Directorate General for Health and Consumer Protection (DG SANCO), was conducted to evaluate the effectiveness of a liquid chromatography/tandem mass spectrometry (LC/MS/MS) method for the determination of semicarbazide (SEM) in different types of baby food at a possible future European regulatory limit (10 ng/g). The test portion of the sample was extracted with hydrochloric acid, and the analyte was derivatized with 2-nitrobenzaldehyde, with 1,2-[15N2, 13C] SEM as an internal standard. The extract was neutralized and then purified on a solid-phase extraction cartridge. The SEM was determined by reversed-phase LC with detection by MS/MS. Apple puree, rice pudding, and meat/vegetable meal baby food materials, spiked with SEM at levels of about 3, 10, and 30 ng/g, respectively, were sent to 20 laboratories in 12 different European countries, which submitted results from 17 participants. Recoveries ranged from 88.8 to 106.1%. Based on results for spiked samples (blind pairs at 3 levels), the relative standard deviations for repeatability (RSDr) ranged from 4.2 to 6.9% and the relative standard deviations for reproducibility (RSDR) ranged from 16.6 to 24.3%. The method showed acceptable within- and between-laboratory precision for all 3 matrixes, as evidenced by HorRat values, at the target levels for the determination of SEM.