Development, validation and comparison of three LC-MS/MS methods for determination of endogenous striatal oleoyl ethanolamine in mice.

Obesity has become a severe public health problem worldwide. An endogenous fatty acid ethanolamine oleoyl ethanolamine (OEA) is reported to be capable of reducing body weight and food intake by increasing striatal extracellular dopamine concentration. However, association between obesity and striatal OEA level remains unknown. As such, it is necessary to develop a sensitive and reliable method to quantitate OEA concentration in striatum. Because true endogenous analytes free blank matrix is not available, surrogate analyte, surrogate matrix and background subtraction methods are often employed for the analysis of endogenous compounds. In this study, three liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods were developed and validated for the determination of OEA concentration in mouse brain homogenate. Interestingly, stability results found that OEA-d4 degraded in brain homogenate under room temperature, while OEA level remarkably increased with time. Since lowering temperature could observably decelerate the endogenous transformation of OEA, sample collection and preparation were carried out under ice-bath condition. Hexane: isopropanol (9:1, v/v) was employed as an extractant for liquid-liquid extraction. After method validation, three methods were applied to quantify OEA in striatum homogenate from C57B6/L mice following normal and high fat diet feeding for 4 months. Results from three methods all showed the striatal OEA level in obesity group was significantly higher than control group and obesity-resist group, which indicated that obesity might be associated with elevated striatal OEA level.

The ethanol extract of Involcucrum castaneae ameliorated ovalbumin-induced airway inflammation and smooth muscle thickening in guinea pigs.

ETHNOPHARMACOLOGICAL RELEVANCE: Involucrum castaneae(IC)is used in Chinese folk medicine to treat various lung diseases, as well as for its reducing phlegm and anti-inflammatory properties. AIM OF THE STUDY: The purpose of this experiment is to verify the effect of IC on airway inflammation, responsiveness in ovalbumin (OVA)-induced asthmatic guinea pigs. The main chemical components of IC were also analyzed. MATERIALS AND METHODS: The potential of the ethanol extract of Involucrum castaneae (EEIC) to protect against OVA-induced allergic airway response in guinea pigs was investigated. The latency of asthma in guinea pigs were recorded after the allergic asthma induced. Enzyme-linked immunosorbent assay (ELISA) was used to measure the levels of immunoglobulin E (IgE), interleukin-5 (IL-5), nerve growth factor (NGF) and interferon-γ (IFN-γ) in asthma allergy. Reverse transcription-PCR (RT-PCR) was used to detect the expression of IL-5 mRNA in asthmatic guinea pig lungs. Paraffin sections of lung tissue were used to analyze pathological changes. The total flavonoid content was determined and the chemical components were analyzed by LC-MS/MS. RESULTS: It was found that EEIC was able to reduce the number of eosinophil (EOS) in bronchoalveolar lavage fluid (BALF) and peripheral blood (PB) in the guinea pig model of OVA -induced asthma. Meanwhile, it also significantly reduced the levels of inflammation-related factors IgE and IL-5, decreased the expression of IL-5 mRNA in lung tissue, and increased the level of IFN-γ. Pathological examination of paraffin section of lung tissue showed that EEIC can reduce the thickening of bronchial smooth muscle and reduce the infiltration damage of tissues by various inflammatory cells. The presence of flavonoids, terpenoids and phenolic compounds in EEIC might be responsible for these activities. CONCLUSION: IC alleviated airway inflammation and smooth muscle thickening in guinea pigs with OVA-sensitized allergic asthma. The paper explains the traditional efficacy and material basis of IC and lays a foundation for further development.

Anti-arthritic activities of ethanol extracts of Circaea mollis Sieb. & Zucc. (whole plant) in rodents.

ETHNOPHARMACOLOGICAL RELEVANCE: Circaea mollis Sieb. & Zucc., a genus of Circaea that follows Onagraceae, has been used for centuries as a folk herb in traditional Chinese medicine (TCM) and Hani Ethnopharmacy for the treatment of joint swelling and pain in rheumatoid arthritis. AIM OF THE STUDY: This study was designed to confirm anti-arthritic effects and its underlying mechanism of ethanol extracts of Circaea mollis Sieb. & Zucc. (EEC), which may contribute to provide the pharmacological basis in the treatment of rheumatoid disease. MATERIALS AND METHODS: Dimethylbenzene (DMB)-induced inflammatory swelling model, hot-plate pain model in mice and Freund's complete adjuvant (FCA)-induced arthritis model in rats were used to evaluate the anti-arthritis effect of EEC. Arthritis severity was done by measuring inflammatory swelling, pain threshol, paw swelling, arthritis index, body weight, spleen index and thymus index. The levels of TNF-α, IL-1ß and IL-10 in sera were measured using ELISA. The pathological change of the ankle joint was also done. Phenolic composition of EEC was analyzed. RESULTS: EEC inhibited inflammatory swelling and increased heat-induced pain threshold in mice. Furthermore, EEC significantly alleviated paw swelling and arthritis index, decreasing the spleen index and thymus index. Besides, EEC down-regulated the serum TNF-α and IL-1ß, and increased the production of serum IL-10 in FCA-induced rats. Histopathological examination demonstrated that EEC can effectively relieve synovial hyperplasia, control the infiltration of the inflammatory and protect cartilage from destruction. CONCLUSION: Our work demonstrated that EEC possessed the potential therapeutic effect against arthritis in rodents which was attributed to modulating proinflammatory cytokines TNF-α, IL-1ß and anti-inflammatory factors IL-10. Flavonoids and polyphenols may contribute to the therapeutic effect of EEC on arthritis.

Accurate determination of a novel vasodilatory ß-blocker TJ0711 using LC-MS/MS: Resolution of an isobaric metabolite interference in dog plasma.

A rapid, robust and sensitive liquid chromatography-tandem mass spectrometry method was developed and validated for bioanalysis of TJ0711, a novel vasodilatory ß-blocker in dog plasma. This assay is able to chromatographically separate TJ0711 from its isobaric metabolite as well as glucuronide conjugates. Chromatographic separation was achieved on a Welch Ultimate-XB C18 column (2.1 × 100 mm, 3 µm). The analyte and internal standard (propranolol) were extracted from plasma by liquid-liquid extraction using ethyl acetate. The mass spectrometric detection was carried out in positive ion multiple reaction monitoring mode. Good linearity was obtained over the concentration range of 0.5-500 ng/mL (r > 0.99) for TJ0711. Moreover, the method had good accuracy (RE ranging from -2.70 to -0.32%) and precision (RSD < 7.55%). TJ0711 was stable in dog plasma for at least 6 h at ambient temperature, for at least 30 days at -20°C and after three freeze-thaw cycles. This method was successfully applied to a preclinical pharmacokinetic study and the results demonstrated linear pharmacokinetics of TJ0711 over a dose range from 0.03 to 0.3 mg/kg. No significant gender differences were observed in TJ0711 plasma pharmacokinetic parameters.

Enantioselective determination of 1-[4-(2-methoxyethyl)phenoxy]-3-[2-(2-methoxyphenoxy)ethylamino]-2-propanol hydrochloride, a novel antihypertensive agent, in rat plasma and tissues by liquid chromatography-tandem mass spectrometry.

Enantioselective biodistribution studies of 1-[4-(2-methoxyethyl)phenoxy]-3-[2-(2-methoxyphenoxy)ethylamino]-2-propanol hydrochloride (TJ0711), a novel antihypertensive agent, require the accurate and precise quantification of each TJ0711 enantiomer in biological fluids and tissues. Here we report a simple and sensitive liquid chromatography with tandem mass spectrometry method for simultaneous determination of (R)-TJ0711 and (S)-TJ0711 in rat plasma and tissue samples using protein precipitation. The influence of column type, temperature, mobile phase composition, and flow rate on the retention and enantioselectivity was evaluated. The separation of the TJ0711 enantiomers was ultimately achieved on a SUMICHIRAL OA-2500 column in 15 min using isocratic elution with ethanol/hexane (40:60) at a flow rate of 0.8 mL/min. Good linearities of spiked analyte concentration from 5 to 2000 ng/mL were achieved and the correlation coefficients (R) were greater than 0.99. The intra- and inter-day accuracy and precision for both analytes were <15% at all concentration levels, and the extraction recoveries were consistent among the five quality control concentrations. This assay was successfully applied to quantify plasma and tissue concentrations of TJ0711 enantiomers in a preclinical study.