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Summary

Dithiocarbamates fungicides (DTCs) are worldwidely used fungicides. Residue analytical methods on DTCs are usually based on headspace gas chromatography, which are not much stable and precise. In this study, a specific, simple and reliable method for determining DTCs fungicides residues was optimized and validated. The DTCs in foods and soils were extracted with an alkaline solution of EDTA and l-cysteine, followed by pH adjusting and methyl derivatization in methyl iodine solution. The organic layer of the reactants was separated, concentrated under vacuum and reconstructed in acetonitrile. DTCs residues were eluted on a C18 column and detected by HPLC-DAD at 272 nm. The S-alkyl derivatives of thiram, mancozeb and propineb were separated at different retention times. At fortified levels of 0.05 mg/kg to 2 mg/kg (residue expressed as CS₂, in mg/kg, the same below), it is found that recoveries for DTCs spiked in apple, cucumber, tomato, rice and soil samples ranged from 70.8% to 105.3%, with relative standard deviations (RSD) from 0.6% to 13.7%. Limits of detection (LODs) and quantification (LOQs) ranged from 0.003 to 0.026 mg/kg and from 0.011 to 0.105 mg/kg for various foods and soils. This method was also applied to real sample tests.

Summary

A new HPLC method has been established for determination of 3-monoiodotyrosine (MIT), 3,5-diiodotyrosine (DIT), 3,5-diiodothyronine (T2), 3,3′,5-triiodothyronine (T3), 3,3′,5′-triiodothyronine (rT3), and thyroxine (T4) produced by hydrolysis of iodinated casein with barium hydroxide. The hydrolytic stability of each analyte was evaluated. Iodinated casein was hydrolyzed with saturated barium hydroxide solution for 16 h at 110°C and the barium ions were then removed as barium sulfate. Reversed-phase HPLC was performed on a 2.1 mm × 150 mm, 5 μm particle, C₁₈ column with a mixture of acetonitrile and 0.1% (v/v) formic acid as mobile phase at a flow rate of 0.2 mL min^–1. Acetonitrile was maintained at 5% (v/v) for 5 min and then increased linearly to 50% (v/v) within 35 min. All analytes were quantified by measuring the absorbance at 280 nm. Validation data indicated the method was linear, with regression coefficients (R ²) > 0.998, in the concentration ranges investigated. Sensitivity was adequate—limits of detection (LOD) were 0.04–0.38 μg mL^–1 and limits of quantification (LOQ) were 0.05–0.38 μg mL^–1. Accuracy and precision were acceptable — for all the analytes recovery was 82.0–93.0% and repeatability, as relative standard deviation, was 1.0–3.0%. Hydrolytic stability tests indicated MIT and DIT are much more stable than the other analytes. rT3 was not released directly from iodinated casein but was formed by deiodination of T4 during hydrolysis. The method could be used to identify iodinated casein, to evaluate its activity and quality, and for supervision and regulation of feed additives.

Summary

Radix Isatidis has widely useful activities including anti-virus, anti-bacterial. Tryptanthrin, indigo, and indirubin are active ingredients in R. Isatidis. Response surface methodology (RSM)-optimized infrared-assisted extraction (IRAE) was developed and combined with HPLC for simultaneous determination of tryptanthrin, indigo, and indirubin from R. Isatidis. IRAE were investigated through extraction yields of the three components and optimized by RSM. The optimum conditions were as follows: infrared power of 129 W, solid/liquid ratio of 1:40 g/mL, and irradiation time of 22.5 min. IRAE conditions obtained by RSM were not only accurate, but also had practical value reflecting the expected optimization. Subsequently, this novel IRAE method was evaluated by extraction yield of the components of R. Isatidis samples from different regions. Compared with common extraction methods including maceration extraction (ME), reflux extraction (RE), ultrasound-assisted extraction (UAE), and microwave-assisted extraction (MAE), IRAE showed higher yield with advantages of no limitation of solvent selection, low cost, convenience under optimum extraction conditions. These results suggested the potential of RSM-optimized IRAE for extraction and analysis of the water-/fat-soluble compositions of Chinese herbal medicine. A simple chromatographic separation for simultaneous determination of tryptanthrin, indigo, and indirubin from Chinese herbal medicine R. Isatidis was performed on a C₁₈ column (Diamonsil 150 mm × 4.6 mm i.d., 5 μm) with a mobile phase isocratic consisting of methanol and water at a flow-rate of 0.8 mL min⁻¹. The retention times of tryptanthrin, indigo, and indirubin were 15.4, 31.9, and 58.6 min, respectively. The linear equations were obtained as follows: y = −3094.5744 + 21208.792x for tryptanthrin (R = 0.9998, 0.9–18.0 μg mL⁻¹), y = 4730.0448 + 30180.567x for indigo (R = 0.9997, 0.5–10.0 μg mL⁻¹) and y = −6582.9045 + 67069.312x for indirubin (R = 0.9997, 0.4–8.0 μg mL⁻¹). The result showed that RSM-optimized IRAE was a simple, efficient pretreatment method for the analysis of complex matrix.

A rapid and sensitive ultraperformance liquid chromatography-multiple reaction monitoring-multi-stage/mass spectrometry (UPLC-MRM-MS/MS) method has been developed for simultaneous quantification of salvianolic acid B and tanshinone IIA of salvia tropolone tablets in dog plasma. This was achieved by performing quantification using the MRM acquisition with two channels of MRM-MS/MS and MS full scan for more accuracy qualitative results, and the fragmentation transitions of m/z 295→249, 191 for tanshinone IIA and m/z 297→279, 251 for IS in positive mode, m/z 717→519, 321 for salvianolic acid B and m/z 295→267, 239 for IS in negative mode were selected. The UPLC separation was achieved within 3 min in a single UPLC run. Linear calibration curves were obtained over the concentration range of 10 pg/mL⁻¹ ng/mL for tanshinone IIA and 100 pg/mL⁻¹ for salvianolic acid B. Lower limit of quantitation (LLOQ) was 10 pg/mL and 100 pg/mL for tanshinone IIA and salvianolic acid B, respectively. The inter-day and intra-day precision (relative standard deviation, RSD) in all samples were less than 8.21%, and the recoveries were over 85.9% for both tanshinone IIA and salvianolic acid B. The two channels of MRM with MS full scan approach could provide both qualitative and quantitative results without the need for repetitive analyses and resulted in the reduction of further confirmation experiments and analytical time. The pharmacokinetic study of the two active components of salvia tropolone tablets following oral gavage administration of dogs was thus explored with this method.