[Rapid screening and identification of 167 illegally added medicines in herbal tea by ultra high performance liquid chromatography-electrostatic field orbitrap high resolution mass spectrometry].

An accurate mass database and a method based on ultra high performance liquid chromatography-electrostatic field orbitrap high resolution mass spectrometry (UHPLC-Orbitrap HRMS) were developed. These were applied in the screening and identification of illegally added medicines in herbal tea. Based on investigations, 167 medicines were selected to build an accurate MS database; these medicines included antipyretic analgesics, glucocorticoids, antibiotics, and antihistamines, among other categories. The database was established using Orbitrap HRMS and TraceFinder software. The database carried information on all selected compounds, including the molecular formula, accurate mass of precursor ions and fragment ions, retention time, and mass spectra. The samples were ultrasonically extracted with a 50% (v/v) methanol aqueous solution. The extracted solutions were separated using a Waters XBrigde BEH C18 column (100 mm×2.1 mm, 2.5 µm). As the mobile phases, 0.1% (v/v) formic acid aqueous solution and acetonitrile containing 0.1% (v/v) formic acid were used, with gradient elution. The sample solutions were analyzed by Orbitrap HRMS in the full-scan MS and data-dependent MS/MS acquisition modes (Full MS/dd-MS2). Positive and negative polarity data were simultaneously acquired. Some parameters were optimized to increase the peak intensity and sensitivity of all compounds. The resolutions in the full-MS scan and dd-MS2 scan were set to 70000 and 17500, respectively. In the full-MS mode, scanning was performed in the range of m/z 100 to 1000. In the MS/MS mode, the normalized collision energy (NEC) was set to 20%, 40%, and 60% for each compound. The inclusion list was not used during the measurement, and the dynamic exclusion time was set to 10.0 s. The loop count was set to 5. After acquiring the sample data with these conditions using Orbitrap MS, they were imported into TraceFinder software, through which the sample information was extracted and automatically matched with the information on compounds in the MS database. Screening and identification were conducted by comparing the retention times as well as the exact masses of precursor ions and fragment ions that were experimentally measured. If the errors between the experimentally and theoretically obtained masses of the precursor ions were below 5×10-6 and the deviations in retention times were less than 20 s, then suspicious positive compounds might be identified. Furthermore, if such compounds possess more than one similar fragment ion with a mass tolerance below 5×10-6, and exhibit similar ion distributions in the MS/MS profiles (compared to those in the database), they could be confirmed to be the same. The validation result showed that all compounds had good linear relationships, with correlation coefficients (r) greater than 0.99. Because pefloxacin, norfloxacin, desloratadine, astemizole and clindamycin had background interference, the method was not suitable for their quantification. Following experiments using three spiked concentrations, the recoveries of the rest 162 compounds were found to be in the range of 66.4%-118.1%, and the relative standard deviations (RSDs, n=6), in the range of 0.1%-16.1%. When the limit of detection (LOD) was 0.2 mg/kg, 83 compounds were detected, while when the LOD was 1.0 mg/kg, 167 compounds were detected. All compounds were matched successfully to the standard added sample with the MS database in TraceFinder software. To lower the likelihood of false positive and false negative results, a quality control method was recommended. The method was applied to analyze 245 herbal tea samples, among which 12 positive samples were detected. Thirteen positive compounds were found, including acetaminophen, diclofenac sodium, chlorpheniramine, brompheniramine, dexamethasone, dexamethasone 21-acetate, prednisone, prednisone 21-acetate, metronidazole, erythromycin, ciprofloxacin, amantadine, and dextromethorphan. In particular, amantadine, dextromethorphan, brompheniramine, and ciprofloxacin were newly detected, compared to standard methods. The developed method is rapid and accurate, and will be useful in the high-throughput screening of illegally added medicines in herbal tea.

Human cadaver model as a training tool for enzymatic burn debridement (Nexobrid®): A feasibility study.

OBJECTIVES: To determine the feasibility of using human cadavers to demonstrate enzymatic burn debridement, as a training aid for clinical staff. MATERIAL AND METHODS: A single, fresh-frozen human cadaver was used. Prior consent had been given. Burns were created by flame and scalding. Unburned control sites were also assessed. Nexobrid® enzymatic burn debridement paste was applied to all sites, in adherence to the local clinical protocol for treating burned patients. After removal of Nexobrid®, wounds were assessed to determine if the cadaveric tissue appeared similar to what would be expected in living burned patients and whether the technique could be viable for training of burn care staff. RESULTS: Nexobrid® had a very similar effect upon burned cadaveric skin to what would have been expected in living burned skin. Burns of partial thickness burn depth and full thickness burn depth were debrided and could be clearly identified. CONCLUSIONS: Fresh-frozen human cadaveric tissue is a valid means of provision of training in the technique of enzymatic burn debridement. This finding was unexpected and shows that our understanding of the mechanism of action of Nexobrid® is incomplete.

[Determination of chloramphenicol in propolis and propolis-derived dietary supplements by high performance liquid chromatography-tandem mass spectrometry].

A method for determining chloramphenicol (CAP) in both propolis and propolis-derived dietary supplements was developed by utilizing high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). The flavones in the samples were removed with a lead acetate solution and ammonia, and the fat-soluble interferences, such as beewax and vegetable oils, were removed with n-hexane after the sample dissolved in ethanol. Tert-butyl methyl ether was used as the back-extraction solvent to reduce co-extracting compounds, such as polyethylene glycol 400 (PEG 400) and glycerol, which are common adjuvants of dietary supplements, and some polar interferences. CAP was detected by HPLC-MS/MS and quantified by the internal standard method. The calibration curve showed a good linearity in the range of 0.20-50.0 µg/L. The limits of detection and the limits of quantification were 0.03 and 0.1 µg/kg, respectively. The recoveries in four different matrices at three spiked levels were in the 86.0%-114.4% range with the relative standard deviations from 0.3% to 4.9%. With the advantages of excellent universality, ease of operation, high sensitivity, and strong anti-interference capability, the proposed method was suitable for the determination of CAP in both propolis and propolis-derived dietary supplements.

[Determination of 28 exogenous medicines and endogenous components in herbal drink using high performance liquid chromatography].

A high performance liquid chromatographic method for the determination of 28 exogenous medicines and endogenous components in the herbal drink was developed. The samples were extracted ultrasonically with methanol-water (70:30, v/v), and the extracts were separated in a Thermo Accucore C18 column (100 mm×4.6 mm, 2.6 µm) with methanol-acetonitrile-20 mmol/L ammonium acetate solution (pH 4.2) as the mobile phases by gradient elution. The flow rate was 1.2 mL/min and the column temperature was 35℃. The detection wavelengths were 254 nm and 220 nm. Quantification analysis was performed by the external standard method. The result showed the compounds had a good linear relationship in the range of 1-100 mg/L, and the correlation coefficients (r) were not less than 0.999. The limits of detection (LODs) of the 28 compounds were 1-10 mg/kg in the liquid sample and 20-200 mg/kg in the solid sample. The average recoveries of the 28 compounds in the liquid and solid samples were in the ranges of 88.8%-118.6% and 92.7%-112.3% with the relative standard deviations (RSDs) of 0.1%-6.7% and 0.1%-6.4%, respectively. The method was applied to analyze 456 herbal drink samples, and 55 positive samples were found. The positive rate was 12.1%. The developed method was simple and reliable, and it was suitable for the determination of 28 components in the herbal drink.